The Effect of Preconditioning Strategies on the Adsorption of Model Proteins onto Screen-Printed Carbon Electrodes.

The preconditioning and modification of the supporting electrode surface is an essential step in every biosensor architecture. In particular, when using screen-printed carbon electrodes (SPEs) as inexpensive and convenient disposable sensor substrates, their somewhat lower electrochemical (surface) reproducibility might represent a complex hurdle. Herein, we investigated the effect of selected preconditioning strategies, such as cyclic voltammetric pretreatment, in H2SO4 and H2O2 and plasma pretreatment with a positive and negative glow discharge, which all improved the electrochemical stability of the unmodified SPEs. Furthermore, we studied the influence of preconditioning strategies on the adsorption kinetics of the two most commonly used building blocks for biosensor preparation, i.e., bovine serum albumin (BSA) and protein A. We observed an advantageous effect of all the examined preconditioning strategies for the modification of SPEs with protein A, being the most effective the negative glow discharge. On the other hand, BSA exhibited a more complex adsorption behavior, with the negative glow discharge as the only generally beneficial preconditioning strategy providing the highest electrochemical stability. Protein A revealed a more substantial impact on the electrochemical signal attenuation than BSA considering their same concentrations in the modification solutions. For both BSA and protein A, we showed that the concentrations of 5 and 10 µg mL-1 already suffice for an electrochemically satisfactorily stable electrode surface after 60 min of incubation time, except for BSA at the positive-plasma-treated electrode.

Sodium-Polyacrylate-Based Electrochemical Sensors for Highly Sensitive Detection of Gaseous Phenol at Room Temperature.

The detection of volatile organic compounds with electrochemical gas sensors is still very challenging regarding their sensitivity, selectivity, and operation at room temperature. There is a need for robust, sensitive, inexpensive, and yet easy-to-operate sensors for phenol and other phenolic compounds that function reliably under ambient conditions. Herein, we present a phenol gas sensor based on a combination of a semisolid, alkaline sodium polyacrylate, and commercial screen-printed electrodes. Sodium polyacrylate was employed as a multifunctional sensing material serving as a (i) gel-like electrolyte, (ii) accumulation milieu, and (iii) derivatization medium. Under ambient conditions, the sensor showed excellent sensitivity in the low ppbv (µg m-3) range, a good linear operation in the examined concentration range of 0.1-1.0 ppmv for up to 105 min accumulation, and low sensitivity toward examined interferences. The sensor also indicated a possibility to differentiate between several phenolic compounds based on their oxidation potential. Given its favorable electroanalytical performance, a strong application potential is envisioned in topical fields such as environmental monitoring, cultural heritage preservation, and occupational health and safety.

The in vivo effects of silver nanoparticles on terrestrial isopods, Porcellio scaber, depend on a dynamic interplay between shape, size and nanoparticle dissolution properties.

The present work aims to study the effects that acute exposure to low concentrations of silver nanoparticles (AgNPs) cause in digestive glands of terrestrial isopods (Porcellio scaber). The experiments were designed to integrate different analytical techniques, such as transmission electron microscopy, atomic absorption spectroscopy, proton induced X-ray emission, and Fourier transform IR imaging (FTIRI), in order to gain a comprehensive insight into the process from the AgNPs' synthesis to their interaction with biological tissues in vivo. To this aim, terrestrial isopods were fed with AgNPs having different shapes, sizes, and concentrations. For all the tested conditions, no toxicity at the whole organism level was observed after 14 days of exposure. However, FTIRI showed that AgNPs caused detectable local changes in proteins, lipids, nucleic acids and carbohydrates at the tissue level, to an extent dependent on the interplay of the AgNPs' properties: shape, size, concentration and dissolution of ions from them.

Short-term assessment of cadmium toxicity and uptake from different types of Cd-based Quantum Dots in the model plant Allium cepa L.

We report on the toxicity and bioaccumulation of three different types of Cd-based quantum dots (QDs), dispersed in aqueous medium, for a model plant Allium cepa L. It is believed that encapsulation of nanoparticles should reduce their toxicity and increase their stability in different environments; in this work we studied how QD encapsulation affects their phytotoxicity. Core, core/shell, and core/shell/shell QDs (CdTe, CdTe/ZnS, and CdTe/CdS/ZnS QDs capped by 2-mercaptopropionic acid) were tested and CdCl2 was used as a positive control. After 24-h and 72-h exposure, total Cd content (MCd) and bioaccumulation factors (BAFs) were determined in all parts of A. cepa plants (roots, bulb, shoot), and the total length of the root system was monitored as a toxicity end-point. Measurements of total Cd content versus free Cd2+ content (with Differential Pulse Voltammetry, DPV) in exposure media showed differences in chemical stability of the three QD types. Correspondingly, selected QDs showed different toxicity for A. cepa and different Cd bioaccumulation patterns. CdTe QDs were the most toxic; their effect was similar to CdCl2 due to the release of free Cd2+, which was confirmed by the DPV measurements. Plants exposed to CdTe QDs also bioaccumulated the most Cd among all QD exposure groups. CdTe/ZnS QDs showed no toxicity and very low bioaccumulation of Cd in A. cepa; the main source of measured Cd in the plants were QDs adsorbed on their roots, which was confirmed by fluorescence microscopy. On the contrary, CdTe/CdS/ZnS QD toxicity and bioaccumulation patterns were similar to those of CdTe QDs and pointed to unstable CdS/ZnS shells.

Comparative in vitro genotoxicity study of ZnO nanoparticles, ZnO macroparticles and ZnCl2 to MDCK kidney cells: Size matters.

In the present study, we evaluated the roles that ZnO particle size and Zn ion release have on cyto- and genotoxicity in vitro. The Madin-Darby canine kidney (MDCK) cells were treated with ZnO nanoparticles (NPs), ZnO macroparticles (MPs), and ZnCl2 as a source of free Zn ions. We first tested cytotoxicity to define sub-cytotoxic exposure concentrations and afterwards we performed alkaline comet and cytokinesis-block micronucleus assays. Additionally, the activities of both catalase (CAT) and glutathione S-transferase (GST) were evaluated in order to examine the potential impairment of cellular stress-defence capacity. The amount of dissolved Zn ions from ZnO NPs in the cell culture medium was evaluated by an optimized voltammetric method. The results showed that all the tested zinc compounds induced similar concentration-dependent cytotoxicity, but only ZnO NPs significantly elevated DNA and chromosomal damage, which was accompanied by a reduction of GST and CAT activity. Although Zn ion release from ZnO NPs in cell culture medium was significant, our results show that this reason alone cannot explain the ZnO genotoxicity seen in this experiment. We discuss that genotoxicity of ZnO NPs depends on the particle size, which determines the physical principles of their dissolution and cellular internalisation.

The role of PVP in the bioavailability of Ag from the PVP-stabilized Ag nanoparticle suspension.

We assessed the bioavailability of Ag from Ag nanoparticles (NPs), stabilized with polyvinylpyrrolidone (PVP), to terrestrial isopods which were exposed to 10, 100 and 1000 µg Ag NPs/g of dry food. Different Ag species were determined in the NP suspension that was fed to isopods: (i) total Ag by atomic absorption spectroscopy, (ii) the sum of Ag-PVP complexes and free Ag+ by anodic stripping voltammetry at the bismuth-film electrode, and (iii) free Ag+ by ion-selective potentiometry. The amounts of Ag species in the consumed food were compared to the masses of Ag accumulated in the isopod digestive glands. Our results show that all three Ag species (Ag NPs, Ag-PVP complexes and free Ag+) could be the source of bioaccumulated Ag, but to various degrees depending on the exposure concentration and transformations in the digestive system. We provide a proof that (i) Ag NPs dissolve and Ag-PVP complexes dissociate in the isopod digestive tract; (ii) the concentration of free Ag+ in the suspension offered to the test organisms is not the only measure of bioavailable Ag. The type of NP stabilizer along with the NP transformations in the digestive system needs to be considered in the creation of new computational models of the nanomaterial fate.

A novel approach to the measurement of surfactant parameters in arthropod digestive juices.

In arthropods, the determination of two important parameters of digestive juices, i.e. the total surfactant concentration and the critical micelle concentration (CMC), is challenging due to small sample volumes and low surfactant concentrations. In this work, we report a successful implementation of potentiometric titrations using the surfactant ion-selective electrode (SISE) and the pyrene fluorescence method (PFM) for the determination of the total surfactant concentration and CMC in the digestive juice of terrestrial isopod crustaceans Porcellio scaber. Pooled digestive juice extracts of four (SISE) or two (PFM) animals were used per measurement run. In both cases, digestive juice extracts in 100 µL of deionized water were sufficient for one measurement run. The total surfactant concentration of P. scaber digestive juice was determined to be 9.2 ± 3.5mM and the CMC was approximately 90 µM. Our work presents an important improvement towards easy CMC determination in small volume samples in comparison with the commonly used stalagmometric technique, where much larger sample volumes are usually needed. To date, the total surfactant concentration was not measured in the digestive juices of arthropods other than Homarus vulgaris, Astacus leptodactylus and Cancer pagurus, for which complex separation and analytical techniques were required. Our results obtained by SISE and PFM therefore present the first successful quantification of surfactants and their CMC in small volumes of arthropod digestive juice without prior separation or purification techniques.

FTIR microscopy reveals distinct biomolecular profile of crustacean digestive glands upon subtoxic exposure to ZnO nanoparticles.

Biomolecular profiling with Fourier-Transform InfraRed Microscopy was performed to distinguish the Zn(2+)-mediated effects on the crustacean (Porcellio scaber) digestive glands from the ones elicited by the ZnO nanoparticles (NPs). The exposure to ZnO NPs or ZnCl2 (1500 and 4000 µg Zn/g of dry food) activated different types of metabolic pathways: some were found in the case of both substances, some only in the case of ZnCl2, and some only upon exposure to ZnO NPs. Both the ZnO NPs and the ZnCl2 increased the protein (∼1312 cm(-1); 1720-1485 cm(-1)/3000-2830 cm(-1)) and RNA concentration (∼1115 cm(-1)). At the highest exposure concentration of ZnCl2, where the effects occurred also at the organismal level, some additional changes were found that were not detected upon the ZnO NP exposure. These included changed carbohydrate (most likely glycogen) concentrations (∼1043 cm(-1)) and the desaturation of cell membrane lipids (∼3014 cm(-1)). The activation of novel metabolic pathways, as evidenced by changed proteins' structure (at 1274 cm(-1)), was found only in the case of ZnO NPs. This proves that Zn(2+) are not the only inducers of the response to ZnO NPs. Low bioavailable fraction of Zn(2+) in the digestive glands exposed to ZnO NPs further supports the role of particles in the ZnO NP-generated effects. This study provides the evidence that ZnO NPs induce their own metabolic responses in the subtoxic range.

Growth of a Novel Nanostructured ZnO Urchin: Control of Cytotoxicity and Dissolution of the ZnO Urchin.

The applications of zinc oxide (ZnO) nanowires (NWs) in implantable wireless devices, such as diagnostic nanobiosensors and nanobiogenerators, have recently attracted enormous attention due to their unique properties. However, for these implantable nanodevices, the biocompatibility and the ability to control the behaviour of cells in contact with ZnO NWs are demanded for the success of these implantable devices, but to date, only a few contrasting results from their biocompatibility can be found. There is a need for more research about the biocompatibility of ZnO nanostructures and the adhesion and viability of cells on the surface of ZnO nanostructures. Here, we introduce synthesis of a new nature-inspired nanostructured ZnO urchin, with the dimensions of the ZnO urchin's acicula being controllable. To examine the biocompatibility and behaviour of cells in contact with the ZnO urchin, the Madin-Darby canine kidney (MDCK) epithelial cell line was chosen as an in vitro experimental model. The results of the viability assay indicated that, compared to control, the number of viable cells attached to the surface of the ZnO urchin and its surrounding area were reduced. The measurements of the Zn contents of cell media confirmed ZnO dissolution, which suggests that the ZnO dissolution in cell culture medium could lead to cytotoxicity. A purposeful reduction of ZnO cytotoxicity was achieved by surface coating of the ZnO urchin with poly(vinylidene fluorid-co-hexafluoropropylene) (PVDF-HFP), which changed the material matrix to slow the Zn ion release and consequently reduce the cytotoxicity of the ZnO urchin without reducing its functionality.

Bioavailability of cobalt and iron from citric-acid-adsorbed CoFe2O4 nanoparticles in the terrestrial isopod Porcellio scaber.

The aim of this study was to determine whether citric acid adsorbed onto cobalt ferrite (CoFe2O4) nanoparticles (NPs) influences the bioavailability of their constituents Co and Fe. Dissolution of Co and Fe was assessed by two measures: (i) in aqueous suspension using chemical analysis, prior to application onto the food of test organisms; and (ii) in vivo, measuring the bioavailability in the model terrestrial invertebrate (Porcellio scaber, Isopoda, Crustacea). The isopods were exposed to citric-acid-adsorbed CoFe2O4 NPs for 2 weeks, and tissue accumulation of Co and Fe was assessed. This was compared to pristine CoFe2O4 NPs, and CoCl2 and Fe(III) salts as positive controls. The combined data shows that citric acid enhances free metal ion concentration from CoFe2O4 NPs in aqueous suspension, although in vivo, very similar amounts of assimilated Co were found in isopods exposed to both types of NPs. Therefore, evaluation of the dissolution in suspension by chemical means is not a good predictor of metal assimilation of this model organism; body assimilation of Co and Fe is rather governed by the physiological capacity of P. scaber for the uptake of these metals. Moreover, we propose that citric acid, due to its chelating properties, may hinder the uptake of Co that dissolves from citric-acid-adsorbed CoFe2O4 NPs, if citric acid is present in sufficient quantity.

Cellular internalization of dissolved cobalt ions from ingested CoFe2O4 nanoparticles: in vivo experimental evidence.

With a model invertebrate animal, we have assessed the fate of magnetic nanoparticles in biologically relevant media, i.e., digestive juices. The toxic potential and the internalization of such nanoparticles by nontarget cells were also examined. The aim of this study was to provide experimental evidence on the formation of Co(2+), Fe(2+), and Fe(3+) ions from CoFe2O4 nanoparticles in the digestive juices of a model organism. Standard toxicological parameters were assessed. Cell membrane stability was tested with a modified method for measurement of its quality. Proton-induced X-ray emission and low energy synchrotron radiation X-ray fluorescence were used to study internalization and distribution of Co and Fe. Co(2+) ions were found to be more toxic than nanoparticles. We confirmed that Co(2+) ions accumulate in the hepatopancreas, but Fe(n+) ions or CoFe2O4 nanoparticles are not retained in vivo. A model biological system with a terrestrial isopod is suited to studies of the potential dissolution of ions and other products from metal-containing nanoparticles in biologically complex media.

Upon exposure to Cu nanoparticles, accumulation of copper in the isopod Porcellio scaber is due to the dissolved Cu ions inside the digestive tract.

The fate of nanoparticles in organisms is of significant interest. In the current work, we used a test system with terrestrial isopods (Porcellio scaber) fed with food spiked with Cu NPs or soluble Cu salt for 14 days. Two different doses were used for spiking to yield final concentrations of 2000 and 5000 µg Cu/g dry food. After the exposure period, part of the exposed group of animals was transferred to clean food to depurate. Cu content was analyzed in the digestive glands, gut, and the 'rest' of the body. Similar patterns of (i) assimilated and depurated amounts of Cu, (ii) Cu body distribution, and (iii) effect on isopods feeding behavior were observed regardless of whether the animals were fed with Cu NPs or soluble Cu salt spiked food. Thus, Cu ions and not Cu NPs were assimilated by the digestive gland cells. Solubilization of the Cu NPs applied to the leaves was also analyzed with chemical methods and recombinant Cu-sensing bacteria. The comparison of the in vitro data on solubilization of Cu NPs and in vivo data on Cu accumulation in the animal tissues showed that about 99% of accumulated copper ions was dissolved from ingested Cu NPs in the digestive system of isopods.

Zinc bioaccumulation in a terrestrial invertebrate fed a diet treated with particulate ZnO or ZnCl2 solution.

A number of reports on potential toxicity of nanoparticles are available, but there is still a lack of knowledge concerning bioaccumulation. The aim of this work was to investigate how different sources of zinc, such as uncoated and unmodified ZnO nanoparticles, ZnCl(2) in solution, and macropowder ZnO influence the bioaccumulation of this metal in the terrestrial isopod Porcellio scaber. After exposure to different sources of Zn in the diet, the amount of assimilated Zn in whole body, the efficiency of zinc assimilation, and bioaccumulation factors (BAFs) were assessed. The bioaccumulation potential of Zn was found to be the same regardless of Zn source. The amount of assimilated Zn and BAF were dose-dependent, and Zn assimilation efficiency was independent of exposure concentrations. The Zn assimilation capacity was found to be up to 16% of ingested Zn. It is known that as much as approximately 20% of Zn can be accreted from ZnO particles by dissolution. We conclude that bioaccumulation of Zn in isopods exposed to particulate ZnO depends most probably on Zn dissolution from ZnO particles and not on bioaccumulation of particulate ZnO.