Material-specific binding peptides empower sustainable innovations in plant health, biocatalysis, medicine and microplastic quantification.

Material-binding peptides (MBPs) have emerged as a diverse and innovation-enabling class of peptides in applications such as plant-/human health, immobilization of catalysts, bioactive coatings, accelerated polymer degradation and analytics for micro-/nanoplastics quantification. Progress has been fuelled by recent advancements in protein engineering methodologies and advances in computational and analytical methodologies, which allow the design of, for instance, material-specific MBPs with fine-tuned binding strength for numerous demands in material science applications. A genetic or chemical conjugation of second (biological, chemical or physical property-changing) functionality to MBPs empowers the design of advanced (hybrid) materials, bioactive coatings and analytical tools. In this review, we provide a comprehensive overview comprising naturally occurring MBPs and their function in nature, binding properties of short man-made MBPs (<20 amino acids) mainly obtained from phage-display libraries, and medium-sized binding peptides (20-100 amino acids) that have been reported to bind to metals, polymers or other industrially produced materials. The goal of this review is to provide an in-depth understanding of molecular interactions between materials and material-specific binding peptides, and thereby empower the use of MBPs in material science applications. Protein engineering methodologies and selected examples to tailor MBPs toward applications in agriculture with a focus on plant health, biocatalysis, medicine and environmental monitoring serve as examples of the transformative power of MBPs for various industrial applications. An emphasis will be given to MBPs' role in detecting and quantifying microplastics in high throughput, distinguishing microplastics from other environmental particles, and thereby assisting to close an analytical gap in food safety and monitoring of environmental plastic pollution. In essence, this review aims to provide an overview among researchers from diverse disciplines in respect to material-(specific) binding of MBPs, protein engineering methodologies to tailor their properties to application demands, re-engineering for material science applications using MBPs, and thereby inspire researchers to employ MBPs in their research.

Preparation of a reference material for microplastics in water-evaluation of homogeneity.

Validation of analytical methods for measurements of microplastics (MP) is severely hampered because of a general lack of reference materials, RM. There is a great need to develop such reference materials. This study presents a concept of three-component kit with immobilised MP in solid NaCl, a surfactant and clean water that can be applied for the production of many types of MP RMs. As proof of concept, an RM for polyethylene terephthalate (PET) particles in water was prepared and evaluated for its homogeneity. The particles ranged from 30 µm (Feretmin) to about 200 µm adapted by wet sieving. A specific number of PET particles were immobilized in about 0.29 g of solid NaCl by freeze-drying 1 mL of a NaCl suspension. By using manual and automated counting, twenty reconstituted 1-L water samples were evaluated for homogeneity with respect to number of PET particles from 30 µm to > 200 µm/L of water. The number of particles was 730 ± 120 (mean ± one standard deviation (SD); n = 10) and 865 ± 155 particles (n = 10) obtained by optical microscopy in two independent laboratories. This corresponded to relative SDs of 16.4 and 17.9% and a mean of 797 ± 151 particles (18.9% RSD, for n = 20). Homogeneity studies of the NaCl carrier without reconstitution resulted in 794 ± 60 particles (7.5% RSD). The homogeneity of PET in the salt carrier was also evaluated directly with respect to mass of PET per vial using an ultra-micro balance. An average mass of 293 ± 41 µg of PET was obtained (14, % RSD for n = 14). Micrographs were recorded to demonstrate the absence of major sources of contamination of the RM components. Information about the particle size distribution and particle shapes was obtained by laser diffraction (LD) and dynamic image analysis (DIA). In addition, the identity of the PET polymer was confirmed by Raman and FT-IR spectroscopy. The RM was developed for a large-scale inter-laboratory comparison of PET particles in water (ILC). Based on the homogeneity results, the material was found to be sufficiently homogeneous to be of meaningful use in the ILC. In a 3-day process, more than 500 samples of PET particles in the NaCl carrier were prepared with good potential for further upscaling with respect to the number of vials or with other kinds of polymers. The stability of PET was not evaluated but it was deemed to be stable for the duration of the ILC.

Analysis of microplastics in drinking water and other clean water samples with micro-Raman and micro-infrared spectroscopy: minimum requirements and best practice guidelines.

Microplastics are a widespread contaminant found not only in various natural habitats but also in drinking waters. With spectroscopic methods, the polymer type, number, size, and size distribution as well as the shape of microplastic particles in waters can be determined, which is of great relevance to toxicological studies. Methods used in studies so far show a huge diversity regarding experimental setups and often a lack of certain quality assurance aspects. To overcome these problems, this critical review and consensus paper of 12 European analytical laboratories and institutions, dealing with microplastic particle identification and quantification with spectroscopic methods, gives guidance toward harmonized microplastic particle analysis in clean waters. The aims of this paper are to (i) improve the reliability of microplastic analysis, (ii) facilitate and improve the planning of sample preparation and microplastic detection, and (iii) provide a better understanding regarding the evaluation of already existing studies. With these aims, we hope to make an important step toward harmonization of microplastic particle analysis in clean water samples and, thus, allow the comparability of results obtained in different studies by using similar or harmonized methods. Clean water samples, for the purpose of this paper, are considered to comprise all water samples with low matrix content, in particular drinking, tap, and bottled water, but also other water types such as clean freshwater.

Microscopy-based high-throughput assays enable multi-parametric analysis to assess adverse effects of nanomaterials in various cell lines.

Manufactured nanomaterials (MNMs) selected from a library of over 120 different MNMs with varied compositions, sizes, and surface coatings were tested by four different laboratories for toxicity by high-throughput/-content (HT/C) techniques. The selected particles comprise 14 MNMs composed of CeO2, Ag, TiO2, ZnO and SiO2 with different coatings and surface characteristics at varying concentrations. The MNMs were tested in different mammalian cell lines at concentrations between 0.5 and 250 µg/mL to link physical-chemical properties to multiple adverse effects. The cell lines are derived from relevant organs such as liver, lung, colon and the immune system. Endpoints such as viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied. Soluble MNMs, Ag and ZnO, were toxic in all cell types. TiO2 and SiO2 MNMs also triggered toxicity in some, but not all, cell types and the cell type-specific effects were influenced by the specific coating and surface modification. CeO2 MNMs were nearly ineffective in our test systems. Differentiated liver cells appear to be most sensitive to MNMs, Whereas most of the investigated MNMs showed no acute toxicity, it became clear that some show adverse effects dependent on the assay and cell line. Hence, it is advised that future nanosafety studies utilise a multi-parametric approach such as HT/C screening to avoid missing signs of toxicity. Furthermore, some of the cell type-specific effects should be followed up in more detail and might also provide an incentive to address potential adverse effects in vivo in the relevant organ.

Physico-chemical properties of manufactured nanomaterials - Characterisation and relevant methods. An outlook based on the OECD Testing Programme.

Identifying and characterising nanomaterials require additional information on physico-chemical properties and test methods, compared to chemicals in general. Furthermore, regulatory decisions for chemicals are usually based upon certain toxicological properties, and these effects may not be equivalent to those for nanomaterials. However, regulatory agencies lack an authoritative decision framework for nanomaterials that links the relevance of certain physico-chemical endpoints to toxicological effects. This paper investigates various physico-chemical endpoints and available test methods that could be used to produce such a decision framework for nanomaterials. It presents an overview of regulatory relevance and methods used for testing fifteen proposed physico-chemical properties of eleven nanomaterials in the OECD Working Party on Manufactured Nanomaterials' Testing Programme, complemented with methods from literature, and assesses the methods' adequacy and applications limits. Most endpoints are of regulatory relevance, though the specific parameters depend on the nanomaterial and type of assessment. Size (distribution) is the common characteristic of all nanomaterials and is decisive information for classifying a material as a nanomaterial. Shape is an important particle descriptor. The octanol-water partitioning coefficient is undefined for particulate nanomaterials. Methods, including sample preparation, need to be further standardised, and some new methods are needed. The current work of OECD's Test Guidelines Programme regarding physico-chemical properties is highlighted.

Silver Nanoparticles and Metallic Silver Interfere with the Griess Reaction: Reduction of Azo Dye Formation via a Competing Sandmeyer-Like Reaction.

Silver (Ag) is the most common nanomaterial (NM) in consumer products. Much research has been focused on elucidating the potential impact of Ag-containing NMs on human health, e.g., cytotoxicity, genotoxicity, or proinflammatory responses. In the case of proinflammatory responses, a frequently used end point is the induction of nitric oxide (NO), which is indirectly quantified as nitrite (NO2-) with the Griess reaction. After preliminary studies in a macrophage-like cell culture system showed anomalous false negative results in the presence of silver nanoparticles (Ag NPs), we studied the influence of Ag on the detection of NO2- in a cell-free environment. Solutions containing a known concentration of NaNO2 were prepared in H2O, PBS, or complete cell culture medium (CCM) and analyzed using the Griess reaction in the presence of Ag in its metallic or ionic state. In Milli-Q H2O, the impact of salts on the detection was investigated using NaCl and KBr. After completion of the Griess reaction, the samples were analyzed spectrophotometrically or chromatographically. It was found that the presence of metallic but not ionic Ag interfered with the quantification of NO2-. The effect was more pronounced in PBS and H2O containing NaCl or KBr. The chromatographical analysis provided evidence of a competing reaction consuming the intermediate diazonium salt, which is critical to the Griess reaction. These findings demonstrate yet another substantial interference of NMs with a frequently used in vitro assay. If gone unnoticed, this interference might cause false negative results and an impaired hazard assessment of Ag NMs.

Effects of metal-bearing nanoparticles (Ag, Au, CdS, ZnO, SiO2) on developing zebrafish embryos.

Due to the increasing commercialization of consumer and industrial products containing nanoparticles (NPs), an increase in the introduction of these materials into the environment is expected. NP toxicity to aquatic organisms depends on multiple biotic and abiotic factors, resulting in an unlimited number of combinations impossible to test in practice. The zebrafish embryo model offers a useful screening tool to test and rank the toxicity of nanomaterials according to those diverse factors. This work aims to study the acute and sublethal toxicity of a set of metal-bearing NPs displaying different properties, in comparison to that of the ionic and bulk forms of the metals, in order to establish a toxicity ranking. Soluble NPs (Ag, CdS and ZnO) showed the highest acute and sublethal toxicity, with LC50 values as low as 0.529 mg Ag l(-1) for Ag NPs of 20 nm, and a significant increase in the malformation prevalence in embryos exposed to 0.1 mg Cd l(-1) of CdS NPs of ∼4 nm. For insoluble NPs, like SiO2 NPs, acute effects were not observed during early embryo development due to the protective effect of the chorion. But effects on larvae could be expected, since deposition of fluorescent SiO2 NPs over the gill lamella and excretion through the intestine were observed after hatching. In other cases, such as for gold NPs, the toxicity could be attributed to the presence of additives (sodium citrate) in the NP suspension, as they displayed a similar toxicity when tested separately. Overall, the results indicated that toxicity to zebrafish embryos depends primarily on the chemical composition and, thus, the solubility of the NPs. Other characteristics, such as size, played a secondary role. This was supported by the observation that ionic forms of the metals were always more toxic than the nano forms, and bulk forms were the least toxic to the developing zebrafish embryos.

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.

Simultaneous determination of size and quantification of silica nanoparticles by asymmetric flow field-flow fractionation coupled to ICPMS using silica nanoparticles standards.

This work proposes the use of multimodal mixtures of monodispersed silica nanoparticles (SiO2-NPs) standards for the simultaneous determination of size and concentration of SiO2-NPs in aqueous suspensions by asymmetric flow field-flow fractionation (AF4) coupled to inductively coupled plasma mass spectrometry (ICPMS). For such a purpose, suspensions of SiO2-NPs standards of 20, 40, 60, 80, 100, and 150 nm were characterized by transmission electronic microscopy (TEM), centrifugal liquid sedimentation (CLS), dynamic light scattering (DLS) and by measuring the Z-potential of the particles as well as the exact concentration of NPs by offline ICPMS. An online AF4-ICPMS method which allowed the separation of all the different sized SiO2-NPs contained in the mixture of standards was developed and the analytical figures of merit were systematically evaluated. The method showed excellent linearity in the studied concentration range (0.1-25 mg L(-1)), limits of detection between 0.16 and 0.3 mg L(-1) for smaller and greater particles, respectively, besides a satisfactory accuracy. AF4 calibration with particles with identical nature to those to be analyzed, also permitted accurate size determination in a pragmatic way. Similarly, by using prechannel calibration with NPs for mass determination it was possible to overcome common quantification problems associated with losses of material during the separation and size-dependent effects. The proposed methodology was successfully applied to the characterization in terms of size and concentration of aqueous test samples containing SiO2-NPs with monomodal size distributions.

Techniques and Protocols for Dispersing Nanoparticle Powders in Aqueous Media-Is there a Rationale for Harmonization?

Selecting appropriate ways of bringing engineered nanoparticles (ENP) into aqueous dispersion is a main obstacle for testing, and thus for understanding and evaluating, their potential adverse effects to the environment and human health. Using different methods to prepare (stock) dispersions of the same ENP may be a source of variation in the toxicity measured. Harmonization and standardization of dispersion methods applied in mammalian and ecotoxicity testing are needed to ensure a comparable data quality and to minimize test artifacts produced by modifications of ENP during the dispersion preparation process. Such harmonization and standardization will also enhance comparability among tests, labs, and studies on different types of ENP. The scope of this review was to critically discuss the essential parameters in dispersion protocols for ENP. The parameters are identified from individual scientific studies and from consensus reached in larger scale research projects and international organizations. A step-wise approach is proposed to develop tailored dispersion protocols for ecotoxicological and mammalian toxicological testing of ENP. The recommendations of this analysis may serve as a guide to researchers, companies, and regulators when selecting, developing, and evaluating the appropriateness of dispersion methods applied in mammalian and ecotoxicity testing. However, additional experimentation is needed to further document the protocol parameters and investigate to what extent different stock dispersion methods affect ecotoxicological and mammalian toxicological responses of ENP.

Critical experimental evaluation of key methods to detect, size and quantify nanoparticulate silver.

Different analytical techniques, sedimentation flow field fractionation (SdFFF), asymmetrical flow field flow fractionation (AF4), centrifugal liquid sedimentation (CLS) and dynamic light scattering (DLS) have been used to give complementary size information about suspensions of silver nanoparticles (AgNPs) in the size range of 20-100 nm by taking advantage of the different physical principles on which are based. Particle morphology was controlled by TEM (Transmission Electron Microscopy). Both SdFFF and AF4 were able to accurately size all AgNPs; among sedimentation based techniques, CLS underestimated the average sizes of larger samples (70 and 100 nm), but it produced the best separation of bimodal mixtures Ag40/60 and Ag40/70 mix compared to SdFFF. On the contrary, DLS overestimated the average sizes of the smallest samples (20 and 30 nm) and it was unable to deal with bimodal mixtures. Quantitative mass and number particle size distributions were also calculated starting from UV-vis signals and ICP-MS data and the results evaluated as a means to address the issue of determining nanoparticle size distributions as required for implementation of European regulations relating to labeling of nanomaterials in consumer products. The results are discussed in light of possible particle aggregation state, analysis repeatability, size resolution and quantitative recoveries.

Solid-phase microextraction/gas chromatography-mass spectrometry method optimization for characterization of surface adsorption forces of nanoparticles.

A complete characterization of the different physico-chemical properties of nanoparticles (NPs) is necessary for the evaluation of their impact on health and environment. Among these properties, the surface characterization of the nanomaterial is the least developed and in many cases limited to the measurement of surface composition and zetapotential. The biological surface adsorption index approach (BSAI) for characterization of surface adsorption properties of NPs has recently been introduced (Xia et al. Nat Nanotechnol 5:671-675, 2010; Xia et al. ACS Nano 5(11):9074-9081, 2011). The BSAI approach offers in principle the possibility to characterize the different interaction forces exerted between a NP's surface and an organic--and by extension biological--entity. The present work further develops the BSAI approach and optimizes a solid-phase microextraction gas chromatography-mass spectrometry (SPME/GC-MS) method which, as an outcome, gives a better-defined quantification of the adsorption properties on NPs. We investigated the various aspects of the SPME/GC-MS method, including kinetics of adsorption of probe compounds on SPME fiber, kinetic of adsorption of probe compounds on NP's surface, and optimization of NP's concentration. The optimized conditions were then tested on 33 probe compounds and on Au NPs (15 nm) and SiO2 NPs (50 nm). The procedure allowed the identification of three compounds adsorbed by silica NPs and nine compounds by Au NPs, with equilibrium times which varied between 30 min and 12 h. Adsorption coefficients of 4.66 ± 0.23 and 4.44 ± 0.26 were calculated for 1-methylnaphtalene and biphenyl, compared to literature values of 4.89 and 5.18, respectively. The results demonstrated that the detailed optimization of the SPME/GC-MS method under various conditions is a critical factor and a prerequisite to the application of the BSAI approach as a tool to characterize surface adsorption properties of NPs and therefore to draw any further conclusions on their potential impact on health.

Gold nanoparticles downregulate interleukin-1ß-induced pro-inflammatory responses.

Interleukin 1 beta (IL-1ß)-dependent inflammatory disorders, such as rheumatoid arthritis and psoriasis, pose a serious medical burden worldwide, where patients face a lifetime of illness and treatment. Organogold compounds have been used since the 1930s to treat rheumatic and other IL-1ß-dependent diseases and, though their mechanisms of action are still unclear, there is evidence that gold interferes with the transmission of inflammatory signalling. Here we show for the first time that citrate-stabilized gold nanoparticles, in a size dependent manner, specifically downregulate cellular responses induced by IL-1ß both in vitro and in vivo. Our results indicate that the anti-inflammatory activity of gold nanoparticles is associated with an extracellular interaction with IL-1ß, thus opening potentially novel options for further therapeutic applications.

Biochemical and behavioural responses of the endobenthic bivalve Scrobicularia plana to silver nanoparticles in seawater and microalgal food.

Because of their bactericidal effects, Ag nanoparticles (Ag NPs) have promising industrial development but could lead to potential ecological risks. The aim of this study was to examine the uptake and effect of silver (soluble or as lactate Ag NPs of 40 nm) at low concentrations (10 µg L(-1)) in the endobenthic bivalve Scrobicularia plana exposed, for 14 days, directly (water) or via the diet (microalgae). The stability of Ag NPs in seawater was examined using dynamic light scattering. Release of soluble Ag from Ag NPs in the experimental media was quantified by using diffusive gradient in thin film. Bioaccumulation of Ag in bivalves was measured by electrothermal atomic absorption spectrometry. Behavioural and biochemical biomarkers were determined in bivalves. Aggregation of Ag NPs and the release of soluble Ag from Ag NPs were observed in the experimental media. For both forms of Ag, bioaccumulation was much more important for waterborne than for dietary exposure. The response of oxidative stress biomarkers (catalase, glutathion S-transferase, superoxide dismutase) was more important after dietary than waterborne exposure to Ag (soluble and NPs). These defences were relatively efficient since they led to a lack of response of damage biomarkers. Burrowing was not affected for bivalves exposed directly or through the diet to both Ag forms but feeding behaviour was impaired after 10 days of dietary exposure. Since no differences of responses to Ag either soluble or nanoparticulate were observed, it seems that labile Ag released from Ag NPs was mainly responsible for toxicity.

Investigating the Cellular Uptake of Model Nanoplastics by Single-Cell ICP-MS.

A synthetic route to producing gold-doped environmentally relevant nanoplastics and a method for the rapid and high-throughput qualitative investigation of their cellular interactions have been developed. Polyethylene (PE) and polyvinyl chloride (PVC) nanoparticles, doped with ultrasmall gold nanoparticles, were synthesized via an oil-in-water emulsion technique as models for floating and sedimenting nanoplastics, respectively. Gold nanoparticles were chosen as a dopant as they are considered to be chemically stable, relatively easy to obtain, interference-free for elemental analysis, and suitable for bio-applications. The suitability of the doped particles for quick detection via inductively coupled plasma mass spectrometry (ICP-MS), operating in single-cell mode (scICP-MS), was demonstrated. Specifically, the method was applied to the analysis of nanoplastics in sizes ranging from 50 to 350 nm, taking advantage of the low limit of detection of single-cell ICP-MS for gold nanoparticles. As an initial proof of concept, gold-doped PVC and PE nanoplastics were employed to quantify the interaction and uptake of nanoplastics by the RAW 264.7 mouse macrophage cell line, using scICP-MS and electron microscopy. Macrophages were chosen because their natural biological functions would make them likely to internalize nanoplastics and, thus, would produce samples to verify the test methodology. Finally, the method was applied to assess the uptake by CaCo-2 human intestinal cells, this being a more relevant model for humanexposure to those nanoplastics that are potentially available in the food chain. For both case studies, two concentrations of nanoplastics were employed to simulate both standard environmental conditions and exceptional circumstances, such as pollution hotspot areas.

Agglomeration Behavior and Fate of Food-Grade Titanium Dioxide in Human Gastrointestinal Digestion and in the Lysosomal Environment.

In the present study, we addressed the knowledge gaps regarding the agglomeration behavior and fate of food-grade titanium dioxide (E 171) in human gastrointestinal digestion (GID). After thorough multi-technique physicochemical characterization including TEM, single-particle ICP-MS (spICP-MS), CLS, VSSA determination and ELS, the GI fate of E 171 was studied by applying the in vitro GID approach established for the regulatory risk assessment of nanomaterials in Europe, using a standardized international protocol. GI fate was investigated in fasted conditions, relevant to E 171 use in food supplements and medicines, and in fed conditions, with both a model food and E 171-containing food samples. TiO2 constituent particles were resistant to GI dissolution, and thus, their stability in lysosomal fluid was investigated. The biopersistence of the material in lysosomal fluid highlighted its potential for bioaccumulation. For characterizing the agglomeration degree in the small intestinal phase, spICP-MS represented an ideal analytical tool to overcome the limitations of earlier studies. We demonstrated that, after simulated GID, in the small intestine, E 171 (at concentrations reflecting human exposure) is present with a dispersion degree similar to that obtained when dispersing the material in water by means of high-energy sonication (i.e., ≥70% of particles <250 nm).

Plastibodies for multiplexed detection and sorting of microplastic particles in high-throughput.

Sensitive high-throughput analytic methodologies are needed to quantify microplastic particles (MPs) and thereby enable routine monitoring of MPs to ultimately secure animal, human, and environmental health. Here we report a multiplexed analytical and flow cytometry-based high-throughput methodology to quantify MPs in aqueous suspensions. The developed analytic MPs-quantification platform provides a sensitive as well as high-throughput detection of MPs that relies on the material binding peptide Liquid Chromatography Peak I (LCI) conjugated to Alexa-fluorophores (LCIF16C-AF488, LCIF16C-AF594, and LCIF16C-AF647). These fluorescent material-binding peptides (also termed plastibodies) were used to fluorescently label polystyrene MPs, whereas Alexa-fluorophores alone exhibited a negligible background fluorescence. Mixtures of polystyrene MPs that varied in size (500 nm to 5 µm) and varied in labeled populations were analyzed and sorted into distinct populations reaching sorting efficiencies >90 % for 1 × 106 sorted events. Finally, a multiplexed quantification and sorting with up to three plastibodies was successfully achieved to validate that the combination of plastibodies and flow cytometry is a powerful and generally applicable methodology for multiplexed analysis, quantification, and sorting of microplastic particles.

Effects of silver nanoparticles in diatom Thalassiosira pseudonana and cyanobacterium Synechococcus sp.

The aim of the present study was to investigate the effect of silver nanoparticles (AgNP) of different sizes toward two primary producer aquatic species. Thalassiosira pseudonana and Synechococcus sp. have been selected as representative models for the lower trophic organisms in marine and freshwater habitats, respectively. Time-dependent cellular growth was measured upon exposure to both AgNP and silver nitrate (AgNO(3)). In addition, AgNP behavior in freshwater and marine waters has been followed by CPS disc centrifuge, in the time frame of AgNP exposure studies, and the kinetic release of silver from AgNP of different sizes was measured by dialysis and inductively coupled plasma mass spectrometry (ICP-MS). The combination and interpretation of all these data suggest that a shared effect of AgNP and released silver was responsible for the toxicity in both organisms. Furthermore, the toxic effects induced by AgNP exposure in the present study seem to result from a mixture of parameters including aggregated state, size of the AgNP, stability of the preparation, and speciation of the released silver.

Amorphous silica nanoparticles do not induce cytotoxicity, cell transformation or genotoxicity in Balb/3T3 mouse fibroblasts.

Although amorphous silica nanoparticles (aSiO(2)NPs) are believed to be non-toxic and are currently used in several industrial and biomedical applications including cosmetics, food additives and drug delivery systems, there is still no conclusive information on their cytotoxic, genotoxic and carcinogenic potential. For this reason, this work has investigated the effects of aSiO(2)NPs on Balb/3T3 mouse fibroblasts, focusing on cytotoxicity, cell transformation and genotoxicity. Results obtained using aSiO(2)NPs, with diameters between 15 nm and 300 nm and exposure times up to 72 h, have not shown any cytotoxic effect on Balb/3T3 cells as measured by the MTT test and the Colony Forming Efficiency (CFE) assay. Furthermore, aSiO(2)NPs have induced no morphological transformation in Balb/3T3 cells and have not resulted in genotoxicity, as shown by Cell Transformation Assay (CTA) and Micronucleus (MN) assay, respectively. To understand whether the absence of any toxic effect could result from a lack of internalization of the aSiO(2)NPs by Balb/3T3 cells, we have investigated the uptake and the intracellular distribution following exposure to 85 nm fluorescently-labelled aSiO(2)NPs. Using fluorescence microscopy, it was observed that fluorescent aSiO(2)NPs are internalized and located exclusively in the cytoplasmic region. In conclusion, we have demonstrated that although aSiO(2)NPs are internalized in vitro by Balb/3T3 mouse fibroblasts, they do not trigger any cytotoxic or genotoxic effect and do not induce morphological transformation, suggesting that they might be a useful component in industrial applications.

Measuring protein structure and stability of protein-nanoparticle systems with synchrotron radiation circular dichroism.

We measure the structural and stability changes of proteins at nanomolar concentration upon interaction with nanoparticles. Using synchrotron radiation circular dichroism (SRCD), we measure a decrease of 6 °C in the thermal unfolding of human serum albumin upon interaction with silver nanoparticles while this does not happen with gold. The use of SRCD allows measuring critical parameters on protein-nanoparticle interactions, and it will provide experimental data on the relative stability of key biological proteins for nanotoxicology.