Size- and oxidative potential-dependent toxicity of environmentally relevant expanded polystyrene styrofoam microplastics to macrophages.

Expanded polystyrene (EPS), also known as Styrofoam, is a widespread global pollutant, and its lightweight floating property increases its chances of weathering by abrasion and ultraviolet (UV) irradiation, resulting in microplastics. Herein, we investigated the effects of particle size ((1 µm versus 10 µm), UV irradiation (pristine versus UV oxidation), and origin (secondary versus primary) on the toxicity of Styrofoam microplastics. The target cells used in this study were selected based on human exposure-relevant cell lines: differentiated THP-1 cells for macrophages, Caco-2 for enterocytes, HepG2 for hepatocytes, and A549 for alveolar epithelial cells. In the differentiated THP-1 cells, the levels of cytotoxicity and inflammatory cytokines showed size- (1 µm > 10 µm), UV oxidation- (UV > pristine), and origin- (secondary > primary) dependency. Furthermore, the intrinsic oxidative potential of the test particles was positively correlated with cellular oxidative levels and toxicity endpoints, suggesting that the toxicity of Styrofoam microplastics also follows the oxidative stress paradigm. Additionally, all microplastics induced the activation of the pyrin domain-containing protein 3 (NLRP3) inflammasome and the release of interleukin-1ß (IL-1ß). These results imply that weathering process can aggravate the toxicity of Styrofoam microplastics due to the increased oxidative potential and decreased particle size.

Rapid generation of aged tire-wear particles using dry-, wet-, and cryo-milling for ecotoxicity testing.

Strict environmental laws have been enacted to regulate the emission of exhaust particulate matter (PM), which is one of the most hazardous pollutants that reduce air quality and pose a serious risk to the human health. In addition, non-exhaust PM, such as road wear, tire wear, and brake wear debris, is a significant source of airborne pollutants. Road dust less than 100 µm in size may include tire wear particles (TWPs), which are broken down into finer particles with sizes on the order of tens of micrometers because of weathering. TWPs can be transported to water bodies via runoff, potentially contaminating water systems and negatively affecting aquatic ecosystems. Therefore, ecotoxicity tests using reference TWPs are required to investigate the impact of TWPs on the human health and environment. In this study, aged TWPs were produced using dry-, wet-, and cryo-milling methods, and the dispersion stability of TWPs in dechlorinated water was evaluated. Aged TWPs prepared by dry- and wet-milling had an average particle size of 20 µm, whereas pristine TWPs had an irregular shape and average particle size of 100 µm. The capacity of the ball-milling cylinder and excessively long 28-d generation time constrain the amount of aged TWPs that can be produced through conventional milling. In contrast, cryo-milling reduces the particle size of TWPs at the rate of -275.0 µm/d, which is nine times higher than that upon dry- and wet-milling. Dispersed cryo-milled TWPs had a hydrodiameter of 2.02 µm and were more stable in the aqueous phase in relation to the other aged TWPs. The results of this study suggest that cryo-milled TWPs can be used for aquatic exposure assessments as controls for real-world TWPs.

The reactive oxygen species as pathogenic factors of fragmented microplastics to macrophages.

The presence of microplastics in the various food web raised concerns on human health, but little is known about the target cells and mechanism of toxicity of microplastics. In this study, we evaluated the toxicity of microplastics using relevant cell lines to the oral route of exposure. Approximately 100 µm-sized fragment-type polypropylene (PP) and polystyrene (PS) particles were prepared by sieving after pulverization and further applied the accelerated weathering using ultraviolet and heat. Thus, the panel of microplastics includes fresh PP (f-PP), fresh PS (f-PS), weathered PP (w-PP), and weathered PS (w-PS). The spherical PS with a similar size was used as a reference particle. Treatment of all types of PP and PS did not show any toxic effects to the Caco-2 cells and HepG2 cells. However, the treatment of microplastics to THP-1 macrophages showed significant toxicity in the order of f-PS > f-PP > w-PS > w-PP. The weathering process significantly reduced the reactive oxygen species (ROS) generation potential of both microplastics because the weathered microplastics have an increased affinity to bind serum protein which acts as a ROS scavenger. The intrinsic ROS generation potential of microplastics showed a good correlation with the toxicity endpoints including cytotoxicity and pro-inflammatory cytokines in THP-1 macrophages. In conclusion, the results of this study suggest that the target cell type of microplastics via oral administration can be macrophages and the pathogenic factor to THP-1 macrophages is the intrinsic ROS generation potential of microplastics. Nevertheless, the toxic effect of microplastics tested in this study was much less than that of nano-sized particles.

Electrochemical microgap immunosensors for selective detection of pathogenic Aspergillus niger.

Aspergillus niger (A. niger) is a well-known allergenic, harmful fungus in the indoor environment that can cause asthmatic symptoms and atopy. Previous immunosensing approach suffers from an insufficient detection limit, mainly because there are no techniques for target amplification. We report an electrochemical immunosensor that selectively quantifies the A. niger based on the detection of extracellular proteins by using a specific interaction with antibody. The sensor was designed to show a decrease in redox current upon binding of the antigens secreted from A. niger onto an antibody-immobilized surface between the interdigitated electrodes. The extracellular proteins were profiled by LC-MS/MS to identify the antigens existing in the A. niger solution. Since the targets of the sensor are the proteins, its sensitivity and selectivity remain almost intact even after filtration of the spores. It was also found that the use of secretion promoter in the sampling stage greatly improved the sensor's limit of detection (LOD) for the spores. By this, the LOD was lowered by a few orders of magnitude so as to reach the value as low as ~101 spores/mL.

Continuous separation of fungal spores in a microfluidic flow focusing device.

The research of fungi is of great importance in a number of fields, such as environmental and healthcare studies. While there are a large number of optical and molecular methods available for characterization and identification of fungi and their spores, their isolation is still conducted using slow and labor-intensive methods. Here, we develop a microfluidic device for the continuous separation of fungal spores from other eukaryotic cells. The spores were separated through the microfluidic device by expanding pinched flow fractionation (PFF) containing the spores, achieving a spatial separation perpendicular to the flow direction according to the spore size. Further branch flow fractionation (BFF) and co-flow of a Newtonian and viscoelastic fluid were used to enhance the separation performance. Using this microfluidic device, we demonstrated the separation of two different types of fungal spores and further separation of fungal spores from eukaryotic cells with a separation efficiency of above 90%. Compared to the existing conventional methods, our microfluidic flow focusing device requires little manual handling and uses small amounts of samples without any pre-treatment steps of the samples.

Rapid and Sensitive Detection of Aspergillus niger Using a Single-Mediator System Combined with Redox Cycling.

Rapid and sensitive mold detection is becoming increasingly important, especially in indoor environments. Common mold detection methods based on double-mediated electron transfer between an electrode and molds are not highly sensitive and reproducible, although they are rapid and simple. Here, we report a sensitive and reproducible detection method specific to Aspergillus niger ( A. niger), based on a single-mediator system combined with electrochemical-chemical (EC) redox cycling. Intracellular NAD(P)H-oxidizing enzymes in molds can convert electro-inactive hydroxy-nitro(so)arenes into electro-active hydroxy-aminoarenes. Since the membrane and wall of A. niger is well permeable to both a substrate (4-nitro-1-naphthol) and a reduced product (4-amino-1-naphthol) in tris buffer (pH 7.5) solution, the electrochemical signal is increased in the presence of A. niger due to two reactions: (i) enzymatic reduction of the substrate to the reduced product and (ii) electrochemical oxidation of the reduced product to an oxidized product. When a reducing agent (NADH) is present in the solution, the oxidized product is reduced back to the reduced product and then electrochemically reoxidized. This EC redox cycling significantly amplifies the electrochemical signal. Moreover, the background level is low and highly reproducible because the substrate and the reducing agent are electro-inactive at an applied potential of 0.20 V. The calculated detection limit for A. niger in a common double-mediator system consisting of Fe(CN)63- and menadione is ∼2 × 104 colony-forming unit (CFU)/mL, but the detection limit in the single-mediator system combined with EC redox cycling is ∼2 × 103 CFU/mL, indicating that the newly developed single-mediator system is more sensitive. Importantly, the detection method requires only an incubation period of 10 min and does not require a washing step, an electrode modification step, or a specific probe.

Acute toxic effects of zinc oxide nanoparticles on Hydra magnipapillata.

Zinc oxide nanoparticles (ZnO NPs) are increasingly used in various products as coating and additive materials for household goods, personal-care products, and drug delivery systems. Because of their broad applications, the potential risks to nontarget organisms associated with their input into aquatic environments have generated much concern. We investigated the acute toxicity, morphological responses, and potential impact on physiology and metabolism in polyps exposed to spherical ZnO NPs of either 20 nm (ZnO NP20) or 100 nm (ZnO NP100). The median lethal concentrations (LC50) of ZnO NP20 were 55.3, 8.7, and 7.0 µg/mL after exposure for 48, 72, and 96 h, respectively; and those of ZnO NP100 were 262.0, 14.9, and 9.9 µg/mL, respectively. The morphological responses of the hydra polyps to a range of ZnO NP concentrations suggest that ZnO NPs may negatively affect neurotransmission in Hydra. ZnO NPs may also induce abnormal regeneration in the polyps by affecting the expression of several genes related to the Wnt signaling pathway. The presence of ZnO NP20 in the hydra tissue was confirmed with electron microscopy. A Gene Ontology analysis of the genes differentially expressed in hydra polyps after exposure to ZnO NP20 for 12 or 24 h revealed changes in various processes, including cellular and metabolic process, stress response, developmental process, and signaling. A KEGG pathway analysis of hydra polyps after exposure of ZnO NP20 or ZnO NP100 for 12 or 24 h demonstrated various changes, including in the DNA replication and repair, endocytosis, lysosomes, Wnt signaling, and natural killer-cell-mediated cytotoxicity pathways, suggesting the mechanisms that maintain cellular homeostasis in response to ZnO NPs. Progesterone-mediated oocyte maturation was also affected by the ZnO NPs nanoparticles, suggesting that they are potential endocrine disruptors. This study should increase our concern regarding the dispersal of ZnO NPs in aquatic environments.

Novel photochromic infinite coordination polymer particles derived from a diarylethene photoswitch.

A novel infinite coordination polymer (DAE-ICP) based on zinc nitrite and a diarylethene photoswitch, with reversible photochromic properties in solution and the solid state upon applying photostimuli, was synthesized and characterized by FT-IR, EDX, FE-SEM and FE-TEM.

Interactions between the antifungal drug myclobutanil and gold and silver nanoparticles in Penicillium digitatum investigated by surface-enhanced Raman scattering.

Surface-enhanced Raman scattering (SERS) of an antifungal reagent, myclobutanil (MCB), was performed on Au and Ag nanoparticles (NPs) to estimate the drug-release behaviors in fungal cells. A density functional theory (DFT) calculation was introduced to predict a favorable binding site of MCB to either the Ag or Au atom. Myclobutanil was presumed to bind more strongly to Au than to Ag in their most stable, optimized geometries of the N4 atom in its 1,2,4-triazole unit binding to the metal atom. Strong intensities were observed in the Ag SERS spectra only at acidic pH values, whereas the most prominent peaks in the Au SERS spectra of MCB matched quite well with those of 1,2,4-triazole regardless of pH conditions. The Raman spectral intensities of the MCB-assembled Ag and Au NPs decreased after treatment with either potato dextrose agar (PDA) or glutathione (GSH). Darkfield microscopy and confocal SERS were performed to analyze the MCB-assembled metal NPs inside Penicillium digitatum fungal cells. The results suggested that MCB was released from the metal NPs in the intracellular GSH in the fungi because we observed only fungal cell peaks.

Lack of genotoxic potential of ZnO nanoparticles in in vitro and in vivo tests.

The industrial application of nanotechnology, particularly using zinc oxide (ZnO), has grown rapidly, including products such as cosmetics, food, rubber, paints, and plastics. However, despite increasing population exposure to ZnO, its potential genotoxicity remains controversial. The biological effects of nanoparticles depend on their physicochemical properties. Preparations with well-defined physico-chemical properties and standardized test methods are required for assessing the genotoxicity of nanoparticles. In this study, we have evaluated the genotoxicity of four kinds of ZnO nanoparticles: 20nm and 70nm size, positively or negatively charged. Four different genotoxicity tests (bacterial mutagenicity assay, in vitro chromosomal aberration test, in vivo comet assay, and in vivo micronucleus test, were conducted, following Organization for Economic Cooperation and Development (OECD) test guidelines with good laboratory practice (GLP) procedures. No statistically significant differences from the solvent controls were observed. These results suggest that surface-modified ZnO nanoparticles do not induce genotoxicity in in vitro or in vivo test systems.

Detection of the mycotoxin citrinin using silver substrates and Raman spectroscopy.

We detected a trace amount of the mycotoxin citrinin using surface-enhanced Raman scattering (SERS) on silver nanoparticle (Ag NP) surfaces. The SERS substrate on hydrophobic Teflon films was also introduced to observe the citrinin peaks. A broad band at ∼1382cm(-1), which was ascribed to the symmetric carboxylate stretching mode, was observed in addition to an antisymmetric carboxylate stretching mode at ∼1568cm(-1) in the Raman spectra. The spectral feature indicated that citrinin would adsorb on Ag NPs via its carboxylate form. Based on density functional theory (DFT) calculations, vibrational mode analysis was performed to compare the Raman spectra of citrinin. DFT calculations also predicted that a bidentate bridge configuration through O15 and O16 atoms in citrinin would be the most stable on three Ag atoms. After treating with Ag NPs, observation of citrinin peaks was attempted in fungal cells of Penicillium citrinum. This work may provide useful insights into the direct observation of the hazardous citrinin mycotoxin using SERS by understanding its adsorption behaviors on Ag surfaces.

Raman detection of localized transferrin-coated gold nanoparticles inside a single cell.

We investigated the cellular uptake behavior of non-fluorescent metal nanoparticles (NPs) by use of surface-enhanced Raman scattering (SERS) combined with dark-field microscopy (DFM). The uptake of Au NPs inside a single cell could also be identified by DFM first and then confirmed by z-depth-dependent SERS at micrometer resolution. Guided by DFM for the location of Au NPs, an intracellular distribution assay was possible using Raman dyes with unique vibrational marker bands in order to identify the three-dimensional location inside the single cell by obtaining specific spectral features. Au NPs modified by 4-mercaptobenzoic acid (MBA) bearing its -COOH surface functional group were used to conjugate transferrin (Tf) protein using the 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) reaction. The protein conjugation reaction on Au surfaces was examined by means of color change, absorption spectroscopy, and SERS. Our results demonstrate that DFM techniques combined with SERS may have great potential for monitoring biological processes with protein conjugation at the single-cell level.

Photochromic optical memory based on non-destructive IR read-out.

The photochromic material 1-(2,5-dimethylthien-1,1-dioxide-3-yl)-2-(2,5-dimethylthien-3-yl)hexafluorocyclopentene (DMTFO2) undergoes reversible photocyclization and ring opening reactions upon alternate irradiation with UV and visible light, respectively. Upon photocyclization, the closed-ring isomer of DMTFO2 showed a strong IR absorption band at 1532 cm(-1) which was not observed for the open-ring isomer. To understand the IR spectra of DMTFO2, theoretical calculations were carried out using Gaussian 03 program at the B3LYP/6-31G(d) level. We also demonstrated non-destructive readout of photochromic recording by IR spectral difference between the open- and closed-ring isomers of DMTFO2 in film. We found that DMTFO2 exhibited a higher contrast ratio of IR image between open- and closed-ring isomer compared with previously reported 1,2-bis(2,5-dimethylthien-3-yl)perfluorocyclopentene (DMTF6) because of the increased photoconversion yield as well as higher vibrational transition intensity. These findings along with its high fatigue resistance indicate that DMTFO2 could be an excellent candidate for photon-mode RW (re-writable) memory devices based on photochromic recording and non-destructive IR read-out.

Cis-to-trans photoconversion of azobenzene self-assembled monolayers on gold nanoparticle surfaces investigated by Raman spectroscopy.

Cis-trans photoconversion behaviors of 4-dimethylaminoazobenzenecarboxylate (4DAzC) on gold nanoparticle surfaces have been investigated by means of Raman spectroscopy. The vibrational features indicated that both the cis- and trans-conformation should coexist after the adsorption on gold nanoparticle surfaces. After the irradiation of the blue light, the intensities of the cis-form peaks were found to decrease on gold. Our work can demonstrate that Raman spectroscopy can be utilized in studying the photoconversion of azobenzene-containing self-assembled monolayers fabricated on metal surfaces.

Fatigue-resistant photochromic dithienylethenes by controlling the oxidation state.

High fatigue-resistant photochromic dithienylethenes were synthesized by controlling the oxidation state of 1,2-bis(2-methyl-1-benzothiophene-3-yl)perfluorocyclopentene (BTF6) and 1,2-bis(2,5-dimethylthien-3-yl)perfluorocyclopentene (DMTF6).

Highly fluorescent photochromic diarylethene in the closed-ring form.

A highly fluorescent diarylethene in the closed-ring form was synthesized by the oxidation of 1,2-bis(2-methyl-1-benzothiophene-3-yl)perfluorocyclopentene (BTF6).

Effects of Metalation State (Free Base, Mg, Zn, Cd) on Excited-State Energy Transfer in Diarylethyne-Linked Porphyrin Dimers.

The ground- and excited-state properties of two new porphyrin dimers have been examined using static and time-resolved optical techniques. One dimer consists of a zinc porphyrin and a magnesium porphyrin (ZnMgU), and the other dimer consists of a cadmium porphyrin and a free base (Fb) porphyrin (CdFbU). In both arrays, the porphyrins are joined by a diarylethyne linker at one meso position with mesityl groups at the nonlinking meso positions. The rates of photoinduced energy transfer are faster for ZnMgU ((9 ps)(-)(1)) and CdFbU ((15 ps)(-)(1)) than found previously for ZnFbU ((24 ps)(-)(1)) and MgFbU ((31 ps)(-)(1)). Only for CdFbU does the yield of excited-state energy transfer (87%) drop below the near-quantitative (>/=99%) level, and this effect derives solely from competition with a very short inherent lifetime ( approximately 100 ps) of the photoexcited Cd porphyrin. The results further illustrate (1) the efficacy of this dimeric architecture for ultrafast excited-state energy transfer, (2) how molecular/electronic properties can be manipulated to tune photoinduced energy flow in multiporphyrin arrays, and (3) key factors impacting effective inter-porphyrin electronic communication, including porphyrin orbital tuning.