Neutral red retention time assay in determination of toxicity of nanoparticles.

The neutral red retention time (NRRT) assay is useful for detecting decreased lysosomal membrane stability in haemocytes sampled from bivalves, a phenomenon often associated with exposure to environmental pollutants including nanomaterials. Bivalves are popular sentinel species in ecotoxicology and use of NRRT in study of species in the genus Mytilus is widespread in environmental monitoring. The NRRT assay has been used as an in vivo test for toxicity of carbon nanoparticles (Moore MN, Readman JAJ, Readman JW, Lowe DM, Frickers PE, Beesley A. 2009. Lysosomal cytotoxicity of carbon nanoparticles in cells of the molluscan immune system: An in vivo study. Nanotoxicology. 3 (1), 40-45). We here report application of this assay adapted to a microtitre plate format to a panel of metal and metal oxide nanoparticles (2 ppm). This showed that copper, chromium and cobalt nanoparticles are toxic by this criterion while gold and titanium nanoparticles are not. As the former three nanoparticles are often reported to be cytotoxic while the latter two are thought to be non-cytotoxic, these data support use of NRRT as a general in vitro assay in nanotoxicology.

Interfacial characteristics and determination of cohesive and adhesive strength of plasma-coated hydroxyapatite via nanoindentation and microscratch techniques.

We investigate the chemical composition and mechanical properties of plasma-deposited hydroxyapatite on grit-blasted Ti-6Al-4V coupons as models of typical prosthetic hip implants. Nanoindentation is used to extract the mechanical properties of the hydroxyapatite (HA) coating and to evaluate the behavior of the material as a function of distance from the interface. A microscratch technique was used to determine parameters of cohesive and adhesive failure of the material that are critical in determining the functionality of these biomedical devices. This delamination method has not been studied in detail before and is usually considered to be unsuitable because of the thickness of the HA and the roughness of the substrate. However, through cross-section analysis of the scratch test, we can determine the point at which the HA delaminates from the substrate. It was concluded that spallation occurs locally, and there is no evidence of gross spallation, indicating that the coating is well adhered to the substrate.

Toxicity of copper oxide nanoparticles in the blue mussel, Mytilus edulis: a redox proteomic investigation.

Relatively little is known about the fate and effects of nanomaterials even in relatively simple organisms such as Mytilus edulis. Here, copper oxide nanoparticles (CuO NP) are shown to induce dose-dependent toxic effects at the biochemical, physiological and tissue levels in the blue mussel. Stable CuO NP suspensions were sized by differential light scattering and nanoparticle tracking analysis to yield average particle diameters of approximately 100 nm. These were administered to M. edulis, at doses of 400, 700 and 1000 ppb. Ingested copper was predominantly located in the gill tissue with small amounts in digestive gland. Fifteen coomassie-stained spots were excised from two dimensional gel electrophoresis separations of gill tissue extacts and identified by peptide mass fingerprinting. These contained six unique proteins (alpha- and beta-tubulin, actin, tropomyosin, triosephosphate isomerase and Cu-Zn superoxide dismutase). Of these, two spots (actin and triosephosphate isomerase) showed decreased protein thiols while three (alpha-tubulin, tropomyosin and Cu-Zn superoxide dismutase) showed increased carbonylation which is indicative of protein oxidation of cytoskeleton and enzymes in response to CuO NP. The neutral red retention time (NRRT) assay revealed toxicity due to the CuO NPs which was comparable with toxic metal oxide nanoparticles such as chromium and cobalt. In contrast, non-toxic titanium and gold metal oxide nanoparticles gave no NRRT effects at similar NP concentrations. Histology revealed deposition of pigmented brown cells in response to CuO NP, located predominantly along the mantle and gill margin but also lining digestive tubules and some of the sinuses and distributed throughout the connective tissue and in the adductor muscle.

A new methodology for studying nanoparticle interactions in biological systems: dispersing titania in biocompatible media using chemical stabilisers.

We report here a highly successful and original protocol for the dispersion of nanoparticles in biocompatible fluids for in vitro and in vivo studies of the nanoparticle-biology interaction. Titania is chosen as a suitable model as it is one of the priority materials listed by the OECD and small particles of the anatase structure are extensively used as e.g. photocatalysts in solar cells. Consequently, its delivery into the environment and its interaction with biological organisms is unavoidable. Therefore, its biological effect needs to be understood. In this work, we prepared stable nanoparticle dispersions of anatase aggregates using citrate stabilisations between 45 and 55 nm at concentrations of up to 10 mg mL(-1). The optimum pH for this type of suspension was 7, resulting in ζ-potentials of approximately -50 mV. The stabilised aggregates were the subject of dialysis to produce stable dispersions without the chemical stabiliser, thus allowing studies in the absence of potentially toxic chemicals. Different sizing techniques such as Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA) and Differential Centrifuge Sedimentation (DCS) were used to characterise the different suspensions. The results obtained with each of these techniques are compared and a critical analysis of the suitability of each technique is given.

Influence of alumina coating on characteristics and effects of SiO2 nanoparticles in algal growth inhibition assays at various pH and organic matter contents.

Silica nanoparticles (NPs) belong to the industrially most important NP types. In a previous study it was shown that amorphous SiO(2) NPs of 12.5 and 27.0 nm are stable in algal growth inhibition assays and that their ecotoxic effects are related to NP surface area. Here, it was hypothesized and demonstrated that an alumina coating completely alters the particle-particle, particle-test medium and particle-algae interactions of SiO(2) NPs. Therefore, stability and surface characteristics, dissolution, nutrient adsorption and effects on algal growth rate of both alumina coated SiO(2) NPs and bare SiO(2) NPs in OECD algal test medium as a function of pH (6.0-8.6) and natural organic matter (NOM) contents (0-12 mg C/l) were investigated. Alumina coated SiO(2) NPs aggregated in all media and adsorbed phosphate depending on pH and NOM concentration. On the other hand, no aggregation or nutrient adsorption was observed for the bare SiO(2) NPs. Due to their positive surface charge, alumina coated SiO(2) NPs agglomerated with Pseudokirchneriella subcapitata. Consequently, algal cell density measurements based on cell counts were unreliable and hence fluorescent detection of extracted chlorophyll was the preferred method. Alumina coated SiO(2) NPs showed lower toxicity than bare SiO(2) NPs at concentrations ≥46 mg/l, except at pH 6.0. At low concentrations, no clear pH effect was observed for alumina coated SiO(2) NPs, while at higher concentrations phosphate deficiency could have contributed to the higher toxicity of those particles at pH 6.0-6.8 compared to higher pH values. Bare SiO(2) NPs were not toxic at pH 6.0 up to 220 mg/l. Addition of NOM decreased toxicity of both particles. For SiO(2) NPs the 48 h 20% effect concentration of 21.8 mg/l increased 2.6-21 fold and a linear relationship was observed between NOM concentration and effective concentrations. No effect was observed for alumina coated SiO(2) NPs in presence of NOM up to 1000 mg/l. All experiments point out that the alumina coating completely altered NP interactions. Due to the difference in surface composition the SiO(2) NPs, which had the smallest surface area, were more toxic to the alga than the alumina coated SiO(2) NPs. Hence, surface modification can dominate the effect of surface area on toxicity.

Carbon composite microelectrodes: charge percolation and electroanalytical performance.

Microelectrodes based on two different epoxy-graphite composites (Araldite-M/HY5162 and Araldite-PY302-2/HY943) that are compatible with organic solvents have been developed and characterized. The variation in the bulk conductivity with graphite particle loading is described by percolation theory and indicates that the particles interact strongly with one another. The percolation threshold is 52% v/v loading of graphite, and this composite exhibits a bulk conductivity of 15 S m(-1). Microdisk electrodes of 25-microm diameter were produced by first etching a microcavity at the tip of a platinum microelectrode, which was then packed with a composite containing 60% v/v graphite so as to optimize both electrical conductivity and the electrode stability in acetonitrile and methanol solutions. Solution phase voltammetry of ferrocene is nearly ideal, and the responses are dominated by radial diffusion (slow scan rates) and semi-infinite linear diffusion (fast scan rates). The microelectrodes display high signal-to-noise ratios, good sensitivity, and low detection limits. The response times given by the product of the resistance, R, and capacitance, C, are 7.5 x 10(-4) and 1.4 x 10(-1) s for the Araldite M and PY302-2 composites, respectively. Although these response times are significantly slower than those associated with microelectrodes based on carbon fibers or metal wires, they are sufficient for time-resolved electroanalytical applications. The long response times arise from the large composite resistances, 3.1 x 10(11) and 8.3 x 10(11) Omega cm(-2) for Araldite M and PY302-2, respectively. Voltammetry of ferrocene in the absence of deliberately added supporting electrolyte is also reported. Significantly, indistinguishable slopes and intercepts for a calibration curve of peak current vs ferrocene concentration where 2 < [ferrocene] < 50 microM are obtained in the presence and absence of supporting electrolyte.

Carbon composite electrodes: surface and electrochemical properties.

Electrodes based on particulate carbon-epoxy or silicone composites have been formed and characterised using electrochemical methods, scanning electron microscopy and scanning electrochemical microscopy. These composites are rigid, exhibit high electrical conductivity and are stable in organic solvents for prolonged periods. The bulk resistance of the Araldite-M and Araldite-CW2215 based electrodes is low, 130+/-12 and 185+/-15 ohms, respectively. In contrast, the bulk resistance of the silicone based electrodes is 1480+/-112 ohms. The uncompensated resistance of electrochemical cells where the composites act as working electrodes is significantly larger than that expected on the basis of solution resistance alone, i.e., up to 7.5 kohms in the case of the silicone composites. These results are interpreted in terms of the presence of pores within the composite material. The response times of the composite electrodes to changes in the applied potential is between 3.1 and 7.2 ms which, although almost an order of magnitude longer than a comparable glassy carbon electrode, is sufficiently rapid to give useful voltammetric data for scan rates of several V s(-1). Close to ideal reversible cyclic voltammetry is observed for ferrocene under semi-infinite diffusion control for scan rates between 0.01 and 0.1 V s(-1) at the Araldite composites. In contrast, the large resistance associated with the silicone based materials causes quasi-reversible responses to be observed over this range of scan rate. Scan rate dependent cyclic voltammetry and time resolved chronoamperometry responses observed for ferrocene in solution are consistent with those expected for a random array of microelectrodes. Scanning electron microscopy and scanning electrochemical microscopy has been used to image the shape, size and electrochemical activity of the electroactive zones. In the case of Araldite-M, the quality of the electrode surface has been probed by comparing the rate of heterogeneous electron transfer at a composite microelectrode with that found for a carbon fibre electrode. The standard heterogeneous electron transfer rate constant, k degrees , is 6.0+/-0.1 x 10(-3) cm s(-1) for the composite compared to 1.5+/-0.1 x 10(-1) cm s(-1) for the carbon fibre electrode. While the smaller rate constant found for the composite suggests a less pristine surface, k degrees is sufficiently large to support reversible, electron transfer under typical electroanalytical conditions. These fundamental measurements will underpin the development of enzyme based biosensors for use in organic solvents.