Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles.

The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-ß and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-ß and TNF-α measurements.

MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs.

Multi-walled carbon nanotubes (MWCNTs) are an inhomogeneous group of nanomaterials that vary in lengths, shapes and types of metal contamination, which makes hazard evaluation difficult. Here we present a toxicogenomic analysis of female C57BL/6 mouse lungs following a single intratracheal instillation of 0, 18, 54 or 162 µg/mouse of a small, curled (CNT(Small), 0.8 ± 0.1 µm in length) or large, thick MWCNT (CNT(Large), 4 ± 0.4 µm in length). The two MWCNTs were extensively characterized by SEM and TEM imaging, thermogravimetric analysis, and Brunauer-Emmett-Teller surface area analysis. Lung tissues were harvested 24h, 3 days and 28 days post-exposure. DNA microarrays were used to analyze gene expression, in parallel with analysis of bronchoalveolar lavage fluid, lung histology, DNA damage (comet assay) and the presence of reactive oxygen species (dichlorodihydrofluorescein assay), to profile and characterize related pulmonary endpoints. Overall changes in global transcription following exposure to CNT(Small) or CNT(Large) were similar. Both MWCNTs elicited strong acute phase and inflammatory responses that peaked at day 3, persisted up to 28 days, and were characterized by increased cellular influx in bronchoalveolar lavage fluid, interstitial pneumonia and gene expression changes. However, CNT(Large) elicited an earlier onset of inflammation and DNA damage, and induced more fibrosis and a unique fibrotic gene expression signature at day 28, compared to CNT(Small). The results indicate that the extent of change at the molecular level during early response phases following an acute exposure is greater in mice exposed to CNT(Large), which may eventually lead to the different responses observed at day 28.

Design, fabrication and characterization of a low-impedance 3D electrode array system for neuro-electrophysiology.

Recent progress in patterned microelectrode manufacturing technology and microfluidics has opened the way to a large variety of cellular and molecular biosensor-based applications. In this extremely diverse and rapidly expanding landscape, silicon-based technologies occupy a special position, given their statute of mature, consolidated, and highly accessible areas of development. Within the present work we report microfabrication procedures and workflows for 3D patterned gold-plated microelectrode arrays (MEA) of different shapes (pyramidal, conical and high aspect ratio), and we provide a detailed characterization of their physical features during all the fabrication steps to have in the end a reliable technology. Moreover, the electrical performances of MEA silicon chips mounted on standardized connector boards via ultrasound wire-bonding have been tested using non-destructive electrochemical methods: linear sweep and cyclic voltammetry, impedance spectroscopy. Further, an experimental recording chamber package suitable for in vitro electrophysiology experiments has been realized using custom-design electronics for electrical stimulus delivery and local field potential recording, included in a complete electrophysiology setup, and the experimental structures have been tested on newborn rat hippocampal slices, yielding similar performance compared to commercially available MEA equipments.

Advances in the Rapid Diagnostic of Viral Respiratory Tract Infections.

Viral infections are a significant public health problem, primarily due to their high transmission rate, various pathological manifestations, ranging from mild to severe symptoms and subclinical onset. Laboratory diagnostic tests for infectious diseases, with a short enough turnaround time, are promising tools to improve patient care, antiviral therapeutic decisions, and infection prevention. Numerous microbiological molecular and serological diagnostic testing devices have been developed and authorised as benchtop systems, and only a few as rapid miniaturised, fully automated, portable digital platforms. Their successful implementation in virology relies on their performance and impact on patient management. This review describes the current progress and perspectives in developing micro- and nanotechnology-based solutions for rapidly detecting human viral respiratory infectious diseases. It provides a nonexhaustive overview of currently commercially available and under-study diagnostic testing methods and discusses the sampling and viral genetic trends as preanalytical components influencing the results. We describe the clinical performance of tests, focusing on alternatives such as microfluidics-, biosensors-, Internet-of-Things (IoT)-based devices for rapid and accurate viral loads and immunological responses detection. The conclusions highlight the potential impact of the newly developed devices on laboratory diagnostic and clinical outcomes.

Detection of human papilloma viruses using nanostructurated silicon support in microarray technology.

In a typical microarray experiment, DNA is arrayed on a solid substrate as spots, the array being probed with a sample or a capture molecule of interest and the interaction monitored through different detection methods. The present study evaluates the possibility to use micro-array technology to genotype samples with Human Papilloma Viruses (HPV). The performance of DNA microarrays strongly depend on their surface properties. The efficiency of DNA immobilization in terms of sensitivity and specificity is one of the most important step in obtaining a microarray chip for diagnosis of HPV family viruses. Here we report the preparation and evaluation of nano-porous silicon surfaces for HPV detection based on DNA micro-array technique. Two different surfaces based on similar porous structure chemically modified in order to efficiently immobilize ss-DNA specific for HPV viruses were investigate.

Nanostructure and internal strain distribution in porous silicon.

Porous silicon (PS) layers with different degrees of porosity have been fabricated and their nanostructure has been investigated using complementary methods as FE-SEM (field emission scanning electron microscopy), SAXS (small-angle X-ray scattering), and Raman spectroscopy. Correlation of these results with strain analyses is also required for envisaged applications in MEMS technology. Symmetrical and asymmetrical rocking curves obtained by high-resolution X-ray diffraction completed with reciprocal space maps (RSMs) explain the features observed in Raman spectra: the PS film in-depth contains two layers-bulk and highly strained superficial layer, between them being a graded strain layer.

SPR detection of protein enhanced by seedless synthesized gold nanorods.

Surface plasmon resonance (SPR) is a label-free, real-time bio-sensing technique with high potential in the diagnostic area, especially when a signal amplification strategy is used to improve the detection limit. We report here a simple method for enhancing the detection limit of bovine serum albumin (BSA), by attaching gold nanorods (AuNRs). AuNRs were obtained by a seedless synthesis technique and characterized using scanning electron microscopy (SEM), UV-VIS spectroscopy, FT-IR spectroscopy and dynamic light scattering (DLS). Finite element method (FEM) simulations were employed to explore the enhancement of the SPR signal by adding AuNRs on the SPR sensor's metallic layer. SPR spectroscopy was used to analyze the changes in the refractive index brought by the immobilization of unconjugated BSA and BSA modified with AuNRs. The results confirmed that the AuNRs conjugated with the protein increase the SPR signal ~ 10 times, leading to a limit of detection of 1.081 × 10-8 M (0.713 µg/mL).

Point-of-Care Testing-The Key in the Battle against SARS-CoV-2 Pandemic.

The deleterious effects of the coronavirus disease 2019 (COVID-19) pandemic urged the development of diagnostic tools to manage the spread of disease. Currently, the "gold standard" involves the use of quantitative real-time polymerase chain reaction (qRT-PCR) for SARS-CoV-2 detection. Even though it is sensitive, specific and applicable for large batches of samples, qRT-PCR is labour-intensive, time-consuming, requires trained personnel and is not available in remote settings. This review summarizes and compares the available strategies for COVID-19: serological testing, Point-of-Care Testing, nanotechnology-based approaches and biosensors. Last but not least, we address the advantages and limitations of these methods as well as perspectives in COVID-19 diagnostics. The effort is constantly focused on understanding the quickly changing landscape of available diagnostic testing of COVID-19 at the clinical levels and introducing reliable and rapid screening point of care testing. The last approach is key to aid the clinical decision-making process for infection control, enhancing an appropriate treatment strategy and prompt isolation of asymptomatic/mild cases. As a viable alternative, Point-of-Care Testing (POCT) is typically low-cost and user-friendly, hence harbouring tremendous potential for rapid COVID-19 diagnosis.

Nanostructured silicon particles for medical applications.

Porous silicon (PS) which has different properties from the bulk material due to the quantum confinement effects is beside other physical properties (e.g., light emitting) bioactive or even bioresorbable. The aim of this paper is to optimise the experimental conditions for the fabrication of nanostructured Si particles and to find the best methods for attaching on its surface molecules of therapeutic interest. The selective porosification has been performed using (i) a dielectric/metallic masking layer micropatterned with corresponding etching windows; (ii) a controlled diffusion process leading to n-type islands into p-type Si substrate. The PS particles were detached from the Si substrate by switching the electrochemical etching conditions from porosification towards the electropolishing regime. Also, similar results were obtained by fabrication of PS multilayer structures subjected to an additional ultrasonation process. Different organic molecules with antitumoral effect, such as chondroitin sulphate (a sulphated glycosaminoglycan), lactoferrin (globular protein with antimicrobial activity) and N-butyldeoxynojirimycin (an imino sugar that inhibits the growth of the CT-2A brain tumour) were covalently attached on the PS particle surface using 3-aminopropyltriethoxysilane (APTS) molecule as linker. Furthermore, to complete the administration/therapy of drugs, for microparticle targeting and imaging, Fe3O4 nanoparticles were integrated in PS matrix by co-precipitation from a solution of iron salts (Fe3+/Fe2+) in alkaline medium. Microscopic and spectroscopic analyses have been used to characterize the Si microparticles. Tumoral cells were cultivated on the nanostructured PS particles and a significant decrease of the cells density was observed on all investigated samples comparatively with the blank substrate without antitumoral molecules.

Comparative analysis of honey and citrate stabilized gold nanoparticles: In vitro interaction with proteins and toxicity studies.

Gold nanoparticles of comparable size were synthetized using honey mediated green method (AuNPs@honey) and citrate mediated Turkevich method (AuNPs@citrate). Their colloidal behavior in two cell media DMEM and RPMI, both supplemented with 10% FBS, was systematically investigated with different characterization techniques in order to evidence how the composition of the media influences their stability and the development of protein/NP complex. We revealed the formation of the protein corona which individually covers the nanoparticles in RPMI media, like a dielectric spacer according to UV-Vis spectroscopy, while DMEM promotes more abundant agglomerations, clustering together the nanoparticles, according to TEM investigations. In order to evaluate the biological impact of nanoparticles, B16 melanoma and L929 mouse fibroblasts cells were used to carry out the viability assays. Generally, the L929 cells were more sensitive than B16 cells to the presence of gold nanoparticles. Measurements of cell viability, proliferation and apoptotic activities of B16 cells indicated that the effects induced by AuNPs@honey were slightly similar to those induced by AuNPs@citrate, however, the toxic response improved in the L929 fibroblast cells following the treatment with AuNPs@honey within the same concentration range from 1 µg/ml to 15 µg/ml for 48 h. Results showed that honey mediated synthesis generates nanoparticles with reduced toxicity trends depending on the cell type, concentration of nanoparticles and exposure time toward various biomedical applications.

Specific detection of stable single nucleobase mismatch using SU-8 coated silicon nanowires platform.

Novel microarray platform for single nucleotide polymorphisms (SNPs) detection has been developed using silicon nanowires (SiNWs) as support and two different surface modification methods for attaining the necessary functional groups. Accordingly, we compared the detection specificity and stability over time of the probes printed on SiNWs modified with (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde (GAD), or coated with a simpler procedure using epoxy-based SU-8 photoresist. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) were used for comparative characterization of the unmodified and coated SiNWs. The hybridization efficiency was assessed by comprehensive statistical analysis of the acquired data from confocal fluorescence scanning of the manufactured biochips. The high detection specificity between the hybridized probes containing different mismatch types was demonstrated on SU-8 coating by one way ANOVA test (adjusted p value *** < .0001). The stability over time of the probes tethered on SiNWs coated with SU-8 was evaluated after 1, 4, 8 and 21 days of probe incubation, revealing values for coefficient of variation (CV) between 2.4% and 5.6%. The signal-to-both-standard-deviations ratio measured for SU-8 coated SiNWs platform was similar to the commercial support, while the APTES-GAD coated SiNWs exhibited the highest values.

High-performance solid state supercapacitors assembling graphene interconnected networks in porous silicon electrode by electrochemical methods using 2,6-dihydroxynaphthalen.

The challenge for conformal modification of the ultra-high internal surface of nanoporous silicon was tackled by electrochemical polymerisation of 2,6-dihydroxynaphthalene using cyclic voltammetry or potentiometry and, notably, after the thermal treatment (800 °C, N2, 4 h) an assembly of interconnected networks of graphene strongly adhering to nanoporous silicon matrix resulted. Herein we demonstrate the achievement of an easy scalable technology for solid state supercapacitors on silicon, with excellent electrochemical properties. Accordingly, our symmetric supercapacitors (SSC) showed remarkable performance characteristics, comparable to many of the best high-power and/or high-energy carbon-based supercapacitors, their figures of merit matching under battery-like supercapacitor behaviour. Furthermore, the devices displayed high specific capacity values along with enhanced capacity retention even at ultra-high rates for voltage sweep, 5 V/s, or discharge current density, 100 A/g, respectively. The cycling stability tests performed at relatively high discharge current density of 10 A/g indicated good capacity retention, with a superior performance demonstrated for the electrodes obtained under cyclic voltammetry approach, which may be ascribed on the one hand to a better coverage of the porous silicon substrate and, on the other hand, to an improved resilience of the hybrid electrode to pore clogging.

Study of the micro- and nanostructured silicon for biosensing and medical applications.

Emerging applications of porous silicon (PS) lies in its ability to incorporate other materials, such as organic groups, organic and inorganic nanoparticles to form (bio)hybrid systems where each individual constituent may be optimized for a particular function. This paper presents our recent experimental results on the fabrication and applications in biosensing of the porous silicon (PS) based microstructures. We have demonstrated that different morphologies of PS, either as-prepared or coated with gold nanoparticles have an important role in biomolecule detection, due to its large internal surface combined with specific electro-optical properties, being in the same time support for immobilization of complementary biomolecules as well as transducer for biochemical interactions. Therefore, we have investigated the photoluminescence properties of nanoporous Si prepared on different Si micropatterned surfaces comparatively with PS/flat Si in order to develop a new simple and versatile process for biosensor transducer fabrication. Meso- and macro-PS have been investigated for protein immobilization and detection using microarray technique or for DNA biomolecule detection by impedance spectroscopy. Finally, we have demonstrated that macroporous silicon constitutes an appropriate substrate for very sensitive SERS biosensors. RAMAN signal of 11-mercaptoundecanoic acid was investigated on Au/macroporous silicon. Various characterisation techniques have been used, optical and scanning electron microscopy (SEM) to investigate samples morphology, X-ray diffraction for nanoparticle structure, Raman and PL spectroscopy, and laser fluorescence detection for chemical and optical properties analysis and impedance spectroscopy for investigation organic molecule attachment on the Au/PS structures.