Assessing Acinetobacter baumannii virulence and treatment with a bacteriophage using zebrafish embryos.

Acinetobacter baumannii is the leading bacteria causative of nosocomial infections, with high fatality rates, mostly due to their multi-resistance to antibiotics. The capsular polysaccharide (k-type) is a major virulence factor. Bacteriophages are viruses that specifically infect bacteria and have been used to control drug-resistant bacterial pathogens. In particular, A. baumannii phages can recognize specific capsules, from a diversity of >125 that exist. This high specificity demands the in vivo identification of the most virulent A. baumannii k-types that need to be targeted by phage therapy. Currently, the zebrafish embryo has particularly attained interest for in vivo infection modeling. In this study, an A. baumannii infection was successfully established, through the bath immersion of tail-injured zebrafish embryos, to study the virulence of eight capsule types (K1, K2, K9, K32, K38, K44, K45, and K67). The model revealed itself as capable of discerning the most virulent (K2, K9, K32, and K45), middle (K1, K38, and K67), and the less virulent (K44) strains. Additionally, the infection of the most virulent strains was controlled in vivo resorting to the same technique, with previously identified phages (K2, K9, K32, and K45 phages). Phage treatments were able to increase the average survival from 35.2% to up to 74.1% (K32 strain). All the phages performed equally well. Collectively, the results show the potential of the model to not only evaluate virulence of bacteria such as A. baumannii but also assess novel treatments' effectiveness.

Correlative Light, Electron Microscopy and Raman Spectroscopy Workflow To Detect and Observe Microplastic Interactions with Whole Jellyfish.

Many researchers have turned their attention to understanding microplastic interaction with marine fauna. Efforts are being made to monitor exposure pathways and concentrations and to assess the impact such interactions may have. To answer these questions, it is important to select appropriate experimental parameters and analytical protocols. This study focuses on medusae of Cassiopea andromeda jellyfish: a unique benthic jellyfish known to favor (sub-)tropical coastal regions which are potentially exposed to plastic waste from land-based sources. Juvenile medusae were exposed to fluorescent poly(ethylene terephthalate) and polypropylene microplastics (<300 µm), resin embedded, and sectioned before analysis with confocal laser scanning microscopy as well as transmission electron microscopy and Raman spectroscopy. Results show that the fluorescent microplastics were stable enough to be detected with the optimized analytical protocol presented and that their observed interaction with medusae occurs in a manner which is likely driven by the microplastic properties (e.g., density and hydrophobicity).

Single-cell ICP-MS for studying the association of inorganic nanoparticles with cell lines derived from aquaculture species.

The current research deals with the use of single-cell inductively coupled plasma-mass spectrometry (scICP-MS) for the assessment of titanium dioxide nanoparticle (TiO2 NP) and silver nanoparticle (Ag NP) associations in cell lines derived from aquaculture species (sea bass, sea bream, and clams). The optimization studies have considered the avoidance of high dissolved background, multi-cell peak coincidence, and possible spectral interferences. Optimum operating conditions were found when using a dwell time of 50 µs for silver and 100 µs for titanium. The assessment of associated TiO2 NPs by scICP-MS required the use of ammonia as a reaction gas (flow rate at 0.75 mL min-1) for interference-free titanium determinations (measurements at an m/z ratio of 131 from the 48Ti(NH)(NH3)4 adduct). The influence of other parameters such as the number of washing cycles and the cell concentration on accurate determinations by scICP-MS was also fully investigated. Cell exposure trials were performed using PVP-Ag NPs (15 and 100 nm, nominal diameter) and citrate-TiO2 NPs (5, 25, and 45 nm, nominal diameter) at nominal concentrations of 10 and 50 µg mL-1 for citrate-TiO2 NPs and 5.0 and 50 µg mL-1 for PVP-Ag NPs. Results have shown that citrate-TiO2 NPs interact with the outer cell membranes, being quite low in the number of citrate-TiO2 NPs that enters the cells (the high degree of aggregation is the main factor which leads to the aggregates being in the extracellular medium). In contrast, PVP-Ag NPs have been found to enter the cells.

Bioaccumulation of titanium dioxide nanoparticles in green (Ulva sp.) and red (Palmaria palmata) seaweed.

A bioaccumulation study in red (Palmaria palmata) and green (Ulva sp.) seaweed has been carried out after exposure to different concentrations of citrate-coated titanium dioxide nanoparticles (5 and 25 nm) for 28 days. The concentration of total titanium and the number and size of accumulated nanoparticles in the seaweeds has been determined throughout the study by inductively coupled plasma mass spectrometry (ICP-MS) and single particle-ICP-MS (SP-ICP-MS), respectively. Ammonia was used as a reaction gas to minimize the effect of the interferences in the 48Ti determination by ICP-MS. Titanium concentrations measured in Ulva sp. were higher than those found in Palmaria palmata for the same exposure conditions. The maximum concentration of titanium (61.96 ± 15.49 µg g-1) was found in Ulva sp. after 28 days of exposure to 1.0 mg L-1 of 5 nm TiO2NPs. The concentration and sizes of TiO2NPs determined by SP-ICP-MS in alkaline seaweed extracts were similar for both seaweeds exposed to 5 and 25 nm TiO2NPs, which indicates that probably the element is accumulated in Ulva sp. mainly as ionic titanium or nanoparticles smaller than the limit of detection in size (27 nm). The implementation of TiO2NPs in Ulva sp. was confirmed by electron microscopy (TEM/STEM) in combination with energy dispersive X-Ray analysis (EDX).

A novel portable label-free electrochemical immunosensor for ultrasensitive detection of Aeromonas salmonicida in aquaculture seawater.

Infectious diseases caused by Aeromonas salmonicida (A. salmonicida) have a huge impact and produce significant losses in aquaculture and fish farming. Fish pathogen early detection is a critical step for the rapid identification and prevention of these problems. This work presents a novel portable label-free ultrasensitive electrochemical immunosensor for A. salmonicida detection in seawater. It consists of a fluidic integrated electrochemical-cell-chip (ECC) with independent chambers enclosing three electrochemical cells (ECs). Anti-A. salmonicida (AbSalm) antibodies were covalently attached to the gold surface of the microfabricated electrodes and were used for the sensitive detection of A. salmonicida. The antibody-antigen immunoreaction was studied by enzyme-linked immunosorbent assay (ELISA), and the surface functionalization was characterized by using quartz crystal microbalance (QCM), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The performance of the developed immunosensor, in terms of sensitivity, repeatability, and specificity, was also studied. The linear working range varied between 1 and 107 CFU mL-1, with a limit of detection (LOD) as low as 1 CFU mL-1. The suitability of the immunosensor for real sample detection was successfully demonstrated via recovery studies performed in spiked seawater samples. The proposed technology supports the use of low-cost and portable instrumentation that concedes the ultrasensitive, simple, and fast quantification of the A. salmonicida. To the best of our knowledge, this is the first portable sensing system for the detection of A. salmonicida in seawater samples, which provides a promising online monitoring platform for the detection of this bacterium in aquaculture facilities.

Fundamentals of Biosensors and Detection Methods.

Biosensors have a great impact on our society to enhance the life quality, playing an important role in the development of Point-of-Care (POC) technologies for rapid diagnostics, and monitoring of disease progression. COVID-19 rapid antigen tests, home pregnancy tests, and glucose monitoring sensors represent three examples of successful biosensor POC devices. Biosensors have extensively been used in applications related to the control of diseases, food quality and safety, and environment quality. They can provide great specificity and portability at significantly reduced costs. In this chapter are described the fundamentals of biosensors including the working principles, general configurations, performance factors, and their classifications according to the type of bioreceptors and transducers. It is also briefly illustrated the general strategies applied to immobilize biorecognition elements on the transducer surface for the construction of biosensors. Moreover, the principal detection methods used in biosensors are described, giving special emphasis on optical, electrochemical, and mass-based methods. Finally, the challenges for biosensing in real applications are addressed at the end of this chapter.

Correction: A novel microfluidic system for the sensitive and cost-effective detection of okadaic acid in mussels.

Correction for 'A novel microfluidic system for the sensitive and cost-effective detection of okadaic acid in mussels' by Ana Castanheira et al., Analyst, 2021, 146, 2638-2645, DOI: 10.1039/D0AN02092C.

A novel microfluidic system for the sensitive and cost-effective detection of okadaic acid in mussels.

Okadaic acid (OA) is produced by marine dinoflagellates and it can be easily accumulated in shellfish, causing intoxications when consumed by humans. Consequently, there is a need for sensitive, reliable and cost-effective methods to detect OA in real samples. In this work, we developed a novel and affordable microfluidic system to detect OA based on the protein phosphatase 1 inhibition colorimetric assay. This enzyme was immobilized in a microfluidic chamber by physisorption in an alumina sol-gel. The results show good enzyme stability over time when maintained at 4 °C. The developed system was sensitive for OA standard solutions, presenting a limit of detection (LOD) of 11.6 nM over a large linear range (43.4 to 3095.8 nM). Our method revealed an LOD as low as 0.2 µg kg-1 and a linear range between 1.47 and 506 µg kg-1 for extracted mussel matrix, detecting OA concentrations in contaminated mussels much lower than the regulated limit (160 µg kg-1). The enzyme stability and reusability along with the simplicity and low cost associated with microfluidics systems make this method very interesting from a commercial point of view.

Magnetic Hyperthermia for Cancer Treatment: Main Parameters Affecting the Outcome of In Vitro and In Vivo Studies.

Magnetic hyperthermia (MHT) is being investigated as a cancer treatment since the 1950s. Recent advancements in the field of nanotechnology have resulted in a notable increase in the number of MHT studies. Most of these studies explore MHT as a stand-alone treatment or as an adjuvant therapy in a preclinical context. However, despite all the scientific effort, only a minority of the MHT-devoted nanomaterials and approaches made it to clinical context. The outcome of an MHT experiment is largely influenced by a number of variables that should be considered when setting up new MHT studies. This review highlights and discusses the main parameters affecting the outcome of preclinical MHT, aiming to provide adequate assistance in the design of new, more efficient MHT studies.

Covalent Organic Framework Composites: Synthesis and Analytical Applications.

In the recent years, composite materials containing covalent organic frameworks (COFs) have raised increasing interest for analytical applications. To date, various synthesis techniques have emerged that allow for the preparation of crystalline and porous COF composites with various materials. Herein, we summarize the most common methods used to gain access to crystalline COF composites with magnetic nanoparticles, other oxide materials, graphene and graphene oxide, and metal nanoparticles. Additionally, some examples of stainless steel, polymer, and metal-organic framework composites are presented. Thereafter, we discuss the use of these composites for chromatographic separation, environmental remediation, and sensing.

Extraction of Ibuprofen from Natural Waters Using a Covalent Organic Framework.

Ibuprofen is one of the most widely used pharmaceuticals, and due to its inefficient removal by conventional wastewater treatment, it can be found in natural surface waters at high concentrations. Recently, we demonstrated that the TpBD-(CF3)2 covalent organic framework (COF) can adsorb ibuprofen from ultrapure water with high efficiency. Here, we investigate the performance of the COF for the extraction of ibuprofen from natural water samples from a lake, river, and estuary. In general, the complexity of the natural water matrix induced a reduction in the adsorption efficiency of ibuprofen as compared to ultrapure water. The best performance, with over 70% adsorption efficiency, was found in lake water, the sample which featured the lowest pH. According to the theoretical calculations, ibuprofen more favorably interacts with the COF pores in the protonated form, which could partially account for the enhanced adsorption efficiency found in lake water. In addition, we explored the effect of the presence of competing pharmaceuticals, namely, acetaminophen and phenobarbital, on the ibuprofen adsorption as binary mixtures. Acetaminophen and phenobarbital were adsorbed by TpBD-(CF3)2 with low efficiency and their presence led to an increase in ibuprofen adsorption in the binary mixtures. Overall, this study demonstrates that TpBD-(CF3)2 is an efficient adsorbent for the extraction of ibuprofen from natural waters as well.

Evaluation and implementation of commercial antibodies for improved nanoparticle-based immunomagnetic separation and real-time PCR for faster detection of Listeria monocytogenes.

L. monocytogenes continues to be a major health issue in Europe, as well as worldwide. Faster methods, not only for detection, but also for sample preparation are of great interest particularly for this slow-growing pathogen. Immunomagnetic separation has been previously reported to be an effective way to concentrate bacteria, and remove inhibitors. In the present study, different commercial antibodies were evaluated to select the most appropriate one, in order to develop a highly specific method. Additionally, magnetic nanoparticles, instead of microparticles, were selected due to their reported advantages (higher surface-volume ration and faster kinetics). Finally, the separation protocol, with a calculated capture efficiency of 95%, was combined with real-time PCR for highly sensitive detection of the concentrated bacteria. The optimized IMS-qPCR allowed to reduce hands-on time in the sample treatment, without affecting the overall performance of the method as a very low limit of detection was still obtained (9.7 CFU/ 25 g) with values for sensitivity, specificity, accuracy, positive and negative predictive values of 100%, resulting in a kappa index of concordance of 1.00. These results were obtained in spiked food samples of different types (chicken, fish, milk, hard and fresh cheese), further demonstrating the applicability of the optimized methodology presented.

Tailoring Covalent Organic Frameworks To Capture Water Contaminants.

Covalent organic frameworks (COFs) are attractive materials receiving increasing interest in the literature due to their crystallinity, large surface area, and pore uniformity. Their properties can be tailored towards specific applications by judicious design of COF building blocks, giving access to tailor-made pore sizes and surfaces. In this Concept article, developments in the field of COFs that have allowed these materials to be explored for contaminant adsorption are discussed. Strategies to obtain water-stable materials with highly ordered structures and large surface areas are reviewed. Post-synthetic modification approaches, by which pore surfaces can be tuned to target specific contaminants, are described. Recent advances in COF formulations, crucial for future implementation in adsorption devices, are highlighted. At the end, future challenges which need to be addressed to allow for the deployment of COFs for the capture of water contaminants will be discussed.

Adsorption of Pharmaceutical Pollutants from Water Using Covalent Organic Frameworks.

Capture of pharmaceutical pollutants from water was studied using a novel fluorine-bearing covalent organic framework TpBD-(CF3 )2 , which showed ibuprofen adsorption capacity of 119 mg g-1 at neutral pH. This value is further enhanced at pH 2, highlighting the potential of this class of materials to serve as adsorbents even under harsh conditions. The adsorbed pharmaceutical can be recovered from TpBD-(CF3 )2 in high yield, offering the option of recycling both the adsorbent and the pharmaceutical. The high efficiency of ibuprofen capture as compared to other less lipophilic pharmaceuticals suggests that COFs can be pre-designed for selective capture of contaminants.

Hapalindoles from the cyanobacterium fischerella: potential sodium channel modulators.

Hapalindoles make up a large group of bioactive metabolites of the cyanobacterial order Stigonematales. 12-epi-Hapalindole E isonitrile, 12-epi-hapalindole C isonitrile, 12-epi-hapalindole J isonitrile, and hapalindole L from Fischerella are acutely toxic for insect larvae; however, the biochemical targets responsible for the biological activities of hapalindoles are not understood. We describe here the electron impact mass spectra of these four hapalindole congeners; their structures were confirmed by nuclear magnetic resonance spectroscopy. In combination with the presented mass spectra of (15)N-labeled species and their retention times on a gas chromatography capillary column, a rapid and reliable determination should be possible in future research. The bioactivity of these hapalindoles was tested on mammalian cells focusing on their effects in the BE(2)-M17 excitable human neuroblastoma cell line. The fluorescent dye Alamar Blue was applied to monitor cytotoxicity, fura-2 to evaluate changes in the cytosolic calcium concentrations, and bis-oxonol to detect effects on membrane potential. Data showed that the hapalindoles did not affect cell viability of the neuroblastoma cells, even when they were incubated for 72 h. Neither depolarization nor initiation of calcium influx was observed in the cells upon hapalindole treatment. However, the data provide evidence that hapalindoles are sodium channel-modulating neurotoxins. They inhibited veratridine-induced depolarization in a manner similar to that of neosaxitoxin. Our data suggest hapalindoles should be added to the growing number of neurotoxic secondary metabolites, such as saxitoxins and anatoxins, already known in freshwater cyanobacteria. As stable congeners, hapalindoles may be a risk in freshwater ecosystems or agricultural water usage and should therefore be considered in water quality assessment.

New versus naturally aged greenhouse cover films: Degradation and micro-nanoplastics characterization under sunlight exposure.

The understanding of microplastic degradation and its effects remains limited due to the absence of accurate analytical techniques for detecting and quantifying micro- and nanoplastics. In this study, we investigated the release of nanoplastics and small microplastics in water from low-density polyethylene (LDPE) greenhouse cover films under simulated sunlight exposure for six months. Our analysis included both new and naturally aged (used) cover films, enabling us to evaluate the impact of natural aging. Additionally, photooxidation effects were assessed by comparing irradiated and non-irradiated conditions. Scanning electron microscopy (SEM) and nanoparticle tracking analysis (NTA) confirmed the presence of particles below 1 µm in both irradiated and non-irradiated cover films. NTA revealed a clear effect of natural aging, with used films releasing more particles than new films but no impact of photooxidation, as irradiated and non-irradiated cover films released similar amounts of particles at each time point. Raman spectroscopy demonstrated the lower crystallinity of the released PE nanoplastics compared to the new films. Flow cytometry and total organic carbon data provided evidence of the release of additional material besides PE, and a clear effect of both simulated and natural aging, with photodegradation effects observed only for the new cover films. Finally, our results underscore the importance of studying the aging processes in both new and used plastic products using complementary techniques to assess the environmental fate and safety risks posed by plastics used in agriculture.

Bioaccumulation, biodistribution, and transformation of polyvinylpyrrolidone-coated silver nanoparticles in edible seaweeds.

Seaweeds are recognised as a potential eco-friendly food source. However, some species have shown the capacity to bioaccumulate many substances of diverse nature, such as inorganic nanoparticles (NPs), which may have potentially harmful effects on them. Among these NPs, silver nanoparticles (AgNPs) have been used to enhance the antifungal and antibacterial properties of the final consumer products, such as textiles and food packages. Their potential release into the aquatic environment raises significant concern, increasing the probability of interaction with aquatic biota, such as macroalgae. In this work, we investigated the differences in bioaccumulation, biodistribution, and transformation of NPs as a function of seaweed species. We selected polyvinylpyrrolidone-coated silver nanoparticles (PVP-AgNPs) as model NP since they remain colloidally stable in seawater, focusing the study only on single particles and not on aggregates. The study was conducted on two different seaweed species with high commercial interest and value as human food: the red seaweed Palmaria palmata and the green seaweed Ulva fenestrata. Single-particle inductively coupled plasma mass spectroscopy (spICP-MS) analysis showed high and similar bioaccumulation of PVP-AgNPs in both seaweeds, in the range of 109 NPs/g of seaweed. However, electron microscopy with energy-dispersive X-ray analysis demonstrated that their time-dependent distribution and transformation in the algal tissue, mainly dissolution and formation of sulfur-rich corona and/or sulfidation, highly depended on the seaweed type. These results indicate that special attention should be given to the presence and transformation of AgNPs in seaweeds intended for human consumption. Not only the dissolution degree but also the speciation of these NPs could heavily impact their bioaccessibility, bioavailability, biodistribution, and toxicity to humans after ingestion.

Detection of submicron- and nanoplastics spiked in environmental fresh- and saltwater with Raman spectroscopy.

Detection of small plastic particles in environmental water samples has been a topic of increasing interest in recent years. A multitude of techniques, such as variants of Raman spectroscopy, have been employed to facilitate their analysis in such complex sample matrices. However, these studies are often conducted for a limited number of plastic types in matrices with relatively little additional materials. Thus, much remains unknown about what parameters influence the detection limits of Raman spectroscopy for more environmentally relevant samples. To address this, this study utilizes Raman spectroscopy to detect six plastic particle types; 161 and 33 nm polystyrene, < 450 nm and 36 nm poly(ethylene terephthalate), 121 nm polypropylene, and 126 nm polyethylene; spiked into artificial saltwater, artificial freshwater, North Sea, Thames River, and Elbe River water. Overall, factors such as plastic particle properties, water matrix composition, and experimental setup were shown to influence the final limits of detection.

A carboxyl-functionalized covalent organic polymer for the efficient adsorption of saxitoxin.

Saxitoxin (STX), the most widely distributed neurotoxin in marine waters and emerging cyanotoxin of concern in freshwaters, causes paralytic shellfish poisoning in humans upon consumption of contaminated shellfish. To allow for the efficient monitoring of this biotoxin, it is of high importance to find high-affinity materials for its adsorption. Herein, we report the design and synthesis of a covalent organic polymer for the efficient adsorption of STX. Two ß-keto-enamine-based materials were prepared by self-assembly of 2,4,6-triformylphloroglucinol (Tp) with 2,5-diaminobenzoic acid (Pa-COOH) to give TpPa-COOH and with 2,5-diaminotoluene (Pa-CH3) to give TpPa-CH3. The carboxylic acid functionalized TpPa-COOH outperformed the methyl-bearing counterpart TpPa-CH3 by an order of magnitude despite the higher long-range order and surface area of the latter. The adsorption of STX by TpPa-COOH was fast with equilibrium reached within 1 h, and the Langmuir adsorption model gave a calculated maximum adsorption capacity, Qm, of 5.69 mg g-1, making this material the best reported adsorbent for this toxin. More importantly, the prepared TpPa-COOH also showed good reusability and high recovery rates for STX in natural freshwater, thereby highlighting the material as a good candidate for the extraction and pre-concentration of STX from aquatic environments.

Innovative Antibacterial, Photocatalytic, Titanium Dioxide Microstructured Surfaces Based on Bacterial Adhesion Enhancement.

Bacterial colonization and biofilm formation are found on nearly all wet surfaces, representing a serious problem for both human healthcare and industrial applications, where traditional treatments may not be effective. Herein, we describe a synergistic approach for improving the performance of antibacterial surfaces based on microstructured surfaces that embed titanium dioxide nanoparticles (TiO2 NPs). The surfaces were designed to enhance bacteria entrapment, facilitating their subsequent eradication by a combination of UVC disinfection and TiO2 NPs photocatalysis. The efficacy of the engineered TiO2-modified microtopographic surfaces was evaluated using three different designs, and it was found that S2-lozenge and S3-square patterns had a higher concentration of trapped bacteria, with increases of 70 and 76%, respectively, compared to flat surfaces. Importantly, these surfaces showed a significant reduction (99%) of viable bacteria after just 30 min of irradiation with UVC 254 nm light at low intensity, being sixfold more effective than flat surfaces. Overall, our results showed that the synergistic effect of combining microstructured capturing surfaces with the chemical functionality of TiO2 NPs paves the way for developing innovative and efficient antibacterial surfaces with numerous potential applications in the healthcare and biotechnology market.