Automated Patch Clamp for the Detection of Tetrodotoxin in Pufferfish Samples.

Tetrodotoxin (TTX) is a marine toxin responsible for many intoxications around the world. Its presence in some pufferfish species and, as recently reported, in shellfish, poses a serious health concern. Although TTX is not routinely monitored, there is a need for fast, sensitive, reliable, and simple methods for its detection and quantification. In this work, we describe the use of an automated patch clamp (APC) system with Neuro-2a cells for the determination of TTX contents in pufferfish samples. The cells showed an IC50 of 6.4 nM for TTX and were not affected by the presence of muscle, skin, liver, and gonad tissues of a Sphoeroides pachygaster specimen (TTX-free) when analysed at 10 mg/mL. The LOD achieved with this technique was 0.05 mg TTX equiv./kg, which is far below the Japanese regulatory limit of 2 mg TTX equiv./kg. The APC system was applied to the analysis of extracts of a Lagocephalus sceleratus specimen, showing TTX contents that followed the trend of gonads > liver > skin > muscle. The APC system, providing an in vitro toxicological approach, offers the advantages of being sensitive, rapid, and reliable for the detection of TTX-like compounds in seafood.

Evaluation of Toxicity Equivalency Factors of Tetrodotoxin Analogues with a Neuro-2a Cell-Based Assay and Application to Puffer Fish from Greece.

Tetrodotoxin (TTX) is a potent marine neurotoxin involved in poisoning cases, especially through the consumption of puffer fish. Knowledge of the toxicity equivalency factors (TEFs) of TTX analogues is crucial in monitoring programs to estimate the toxicity of samples analyzed with instrumental analysis methods. In this work, TTX analogues were isolated from the liver of a Lagocephalus sceleratus individual caught on South Crete coasts. A cell-based assay (CBA) for TTXs was optimized and applied to the establishment of the TEFs of 5,11-dideoxyTTX, 11-norTTX-6(S)-ol, 11-deoxyTTX and 5,6,11-trideoxyTTX. Results showed that all TTX analogues were less toxic than the parent TTX, their TEFs being in the range of 0.75-0.011. Then, different tissues of three Lagocephalus sceleratus individuals were analyzed with CBA and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The obtained TEFs were applied to the TTX analogues' concentrations obtained by LC-MS/MS analysis, providing an indication of the overall toxicity of the sample. Information about the TEFs of TTX analogues is valuable for food safety control, allowing the estimation of the risk of fish products to consumers.

Detection of Ciguatoxins and Tetrodotoxins in Seafood with Biosensors and Other Smart Bioanalytical Systems.

The emergence of marine toxins such as ciguatoxins (CTXs) and tetrodotoxins (TTXs) in non-endemic regions may pose a serious food safety threat and public health concern if proper control measures are not applied. This article provides an overview of the main biorecognition molecules used for the detection of CTXs and TTXs and the different assay configurations and transduction strategies explored in the development of biosensors and other biotechnological tools for these marine toxins. The advantages and limitations of the systems based on cells, receptors, antibodies, and aptamers are described, and new challenges in marine toxin detection are identified. The validation of these smart bioanalytical systems through analysis of samples and comparison with other techniques is also rationally discussed. These tools have already been demonstrated to be useful in the detection and quantification of CTXs and TTXs, and are, therefore, highly promising for their implementation in research activities and monitoring programs.

Cyclodextrin polymer clean-up method for the detection of ciguatoxins in fish with cell-based assays.

Ciguatoxins (CTXs) are marine toxins produced by microalgae of the genera Gambierdiscus and Fukuyoa, which are transferred through the food webs, reaching humans and causing a poisoning known as ciguatera. The cell-based assay (CBA) is commonly used for their detection because of its high sensitivity and the provided toxicological information. However, matrix effects may interfere in the CBA. In this work, γ-cyclodextrin-hexamethylene diisocyanate (γ-CD-HDI), γ-cyclodextrin-epichlorohydrin (γ-CD-EPI) and γ-CD-EPI conjugated to magnetic beads (γ-CD-EPI-MB) have been evaluated as clean-up materials for fish flesh extracts containing CTXs. The best results were achieved with γ-CD-HDI in column format, which showed a CTX1B recovery of 42% and 32% for Variola louti and Seriola dumerili, respectively, and allowed exposing cells to at least 400 mg/mL of fish flesh. This clean-up strategy provides at least 4.6 and 3.0-fold higher sensitivities to the assay for V.louti and S.dumerili, respectively, improving the reliability of CTX quantification.

Cell immunolocalization of ciguatoxin-like compounds in the benthic dinoflagellate Gambierdiscus australes M. Chinain & M.A. Faust by confocal microscopy.

Dinoflagellates of the genera Gambierdiscus and Fukuyoa are able to produce potent neurotoxins like ciguatoxins (CTXs), which, after biooxidation in fish, are responsible for ciguatera intoxication. An isolate of G. australes from the Canary Islands, that revealed the presence of CTX-like compounds by immunosensing tools, was studied by immunocytochemistry to localize intracellular CTX-like compounds, using 8H4 monoclonal antibody that specifically recognizes the right wing of CTX1B and CTX3C analogues. Confocal microscopy observations of immunostained whole cells revealed a strong positive reaction on cell surface and all along the cell outline, while no reaction was detected inside the cells, probably because the antibody was not able to pass through thecal plates. Cell sections showed a positive antibody staining not only on thecal plates, but also inside cytoplasm, with numerous small dots and larger tubule-like reticulate structures. Small fluorescent dots were detected also on the nuclear surface. These observations indicate that CTX-like compounds are present in G. australes cytoplasm, and then are, at least in part, released to cover the cell surface.

Immobilisation of Neuro-2a cells on electrodes and electrochemical detection of MTT formazan crystals to assess their viability.

Marine toxins are potent toxic compounds that may reach humans and poison them. Therefore, their detection in seafood is crucial to prevent intoxication cases. Colorimetric cell-based assays (CBAs) have been developed to analyse marine neurotoxins, such as ciguatoxins (CTXs) and tetrodotoxins (TTXs), and are based on the toxicological effect of these toxins on the cells. Cell viability can be quantified by measuring the mitochondrial activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). With the purpose of moving forward in the development of cell-based biosensors (CBBs) for neurotoxins, Neuro-2a cells were immobilised on electrodes of different materials (carbon, carbon/polyaniline, carbon/poly-l-lysine, carbon/poly(3,4-ethylenedioxythiophene) and gold) and their presence and viability were assessed by the detection of MTT formazan crystals with cyclic voltammetry (CV). Best results in terms of oxidation potential and current intensity were achieved with carbon and carbon/polyaniline electrodes. Light microscopy also proved the presence of immobilised and living cells on electrodes. Cell density, incubation time and MTT concentration were optimised. Appropriate electrochemical responses were obtained incubating 100,000 cells/electrode for 2 h and using 0.86 mg/mL MTT. The system was able to detect toxicity when exposed to CTX1B and TTX standard solutions as well as Seriola dumerili and Lagocephalus sceleratus fish extracts containing these toxins.

Identification of New CTX Analogues in Fish from the Madeira and Selvagens Archipelagos by Neuro-2a CBA and LC-HRMS.

Ciguatera Poisoning (CP) is caused by consumption of fish or invertebrates contaminated with ciguatoxins (CTXs). Presently CP is a public concern in some temperate regions, such as Macaronesia (North-Eastern Atlantic Ocean). Toxicity analysis was performed to characterize the fish species that can accumulate CTXs and improve understanding of the ciguatera risk in this area. For that, seventeen fish specimens comprising nine species were captured from coastal waters inMadeira and Selvagens Archipelagos. Toxicity was analysed by screening CTX-like toxicity with the neuroblastoma cell-based assay (neuro-2a CBA). Afterwards, the four most toxic samples were analysed with liquid chromatography-high resolution mass spectrometry (LC-HRMS). Thirteen fish specimens presented CTX-like toxicity in their liver, but only four of these in their muscle. The liver of one specimen of Muraena augusti presented the highest CTX-like toxicity (0.270 ± 0.121 µg of CTX1B equiv·kg-1). Moreover, CTX analogues were detected with LC-HRMS, for M. augusti and Gymnothorax unicolor. The presence of three CTX analogues was identified: C-CTX1, which had been previously described in the area; dihydro-CTX2, which is reported in the area for the first time; a putative new CTX m/z 1127.6023 ([M+NH4]+) named as putative C-CTX-1109, and gambieric acid A.

Supramolecular Complexes of Plant Neurotoxin Veratridine with Cyclodextrins and Their Antidote-like Effect on Neuro-2a Cell Viability.

Veratridine (VTD) is a plant neurotoxin that acts by blocking the voltage-gated sodium channels (VGSC) of cell membranes. Symptoms of VTD intoxication include intense nausea, hypotension, arrhythmia, and loss of consciousness. The treatment for the intoxication is mainly focused on treating the symptoms, meaning there is no specific antidote against VTD. In this pursuit, we were interested in studying the molecular interactions of VTD with cyclodextrins (CDs). CDs are supramolecular macrocycles with the ability to form host-guest inclusion complexes (ICs) inside their hydrophobic cavity. Since VTD is a lipid-soluble alkaloid, we hypothesized that it could form stable inclusion complexes with different types of CDs, resulting in changes to its physicochemical properties. In this investigation, we studied the interaction of VTD with ß-CD, γ-CD and sulfobutyl ether ß-CD (SBCD) by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. Docking and molecular dynamics studies confirmed the most stable configuration for the inclusion complexes. Finally, with an interest in understanding the effects of the VTD/CD molecular interactions, we performed cell-based assays (CBAs) on Neuro-2a cells. Our findings reveal that the use of different amounts of CDs has an antidote-like concentration-dependent effect on the cells, significantly increasing cell viability and thus opening opportunities for novel research on applications of CDs and VTD.

An Updated Review of Tetrodotoxin and Its Peculiarities.

Tetrodotoxin (TTX) is a crystalline, weakly basic, colorless organic substance and is one of the most potent marine toxins known. Although TTX was first isolated from pufferfish, it has been found in numerous other marine organisms and a few terrestrial species. Moreover, tetrodotoxication is still an important health problem today, as TTX has no known antidote. TTX poisonings were most commonly reported from Japan, Thailand, and China, but today the risk of TTX poisoning is spreading around the world. Recent studies have shown that TTX-containing fish are being found in other regions of the Pacific and in the Indian Ocean, as well as the Mediterranean Sea. This review aims to summarize pertinent information available to date on the structure, origin, distribution, mechanism of action of TTX and analytical methods used for the detection of TTX, as well as on TTX-containing organisms, symptoms of TTX poisoning, and incidence worldwide.

Electrochemical biosensor for the dual detection of Gambierdiscus australes and Gambierdiscus excentricus in field samples. First report of G. excentricus in the Balearic Islands.

Several genera of marine dinoflagellates are known to produce bioactive compounds that affect human health. Among them, Gambierdiscus and Fukuyoa stand out for their ability to produce several toxins, including the potent neurotoxic ciguatoxins (CTXs), which accumulate through the food web. Once fishes contaminated with CTXs are ingested by humans, it can result in an intoxication named ciguatera. Within the two genera, only some species are able to produce toxins, and G. australes and G. excentricus have been highlighted to be the most abundant and toxic. Although the genera Gambierdiscus and Fukuyoa are endemic to tropical areas, their presence in subtropical and temperate regions has been recently recorded. In this work, the combined use of species-specific PCR primers for G. australes and G. excentricus modified with short oligonucleotide tails allowed the development of a multiplex detection system for these two toxin-producing species. Simultaneous detection was achieved using capture probes specific for G. australes and G. excentricus immobilized on maleimide-coated magnetic beads (MBs), separately placed on the working electrodes of a dual electrode array. Additionally, a rapid DNA extraction technique based on a portable bead beater system and MBs was developed, significantly reducing the extraction time (from several hours to 30 min). The developed technique was able to detect as low as 10 cells of both Gambierdiscus species and allowed the first detection of G. excentricus in the Balearic Islands in 8 out of the 12 samples analyzed. Finally, field samples were screened for CTXs with an immunosensor, successfully reporting 13.35 ± 0.5 pg CTX1B equiv. cell-1 in one sample and traces of toxins in 3 out of the 9 samples analyzed. These developments provide rapid and cost-effective strategies for ciguatera risk assessment, with the aim of guaranteeing seafood safety.

A smartphone-controlled amperometric immunosensor for the detection of Pacific ciguatoxins in fish.

Ciguatoxins (CTXs) are marine neurotoxins produced by microalgae of the genera Gambierdiscus and Fukuyoa. CTXs may reach humans through food webs and cause ciguatera fish poisoning (CFP). An immunosensor for the detection of Pacific CTXs in fish was developed using multiwalled carbon nanotube (MWCNT)-modified carbon electrodes and a smartphone-controlled potentiostat. The biosensor attained a limit of detection (LOD) and a limit of quantification (LOQ) of 6 and 27 pg/mL of CTX1B, respectively, which were 0.001 and 0.005 µg/kg in fish flesh. In the analysis of fish samples from Japan and Fiji, excellent correlations were found with sandwich enzyme-linked immunosorbent assays (ELISAs), a cell-based assay (CBA) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Stability of at least 3 months at -20 °C was predicted. In just over 2 h, the biosensor provides reliable, accurate and precise Pacific CTX contents in fish extracts, being suitable for monitoring and research programs.

Nucleic acid lateral flow dipstick assay for the duplex detection of Gambierdiscus australes and Gambierdiscus excentricus.

The proliferation of harmful microalgae endangers aquatic ecosystems and can have serious economic implications on a global level. Harmful microalgae and their associated toxins also pose a threat to human health since they can cause seafood-borne diseases such as ciguatera. Implementation of DNA-based molecular methods together with appropriate detection strategies in monitoring programs can support the efforts for effective prevention of potential outbreaks. A PCR-lateral flow assay (PCR-LFA) in dipstick format was developed in this work for the detection of two Gambierdiscus species, G. australes and G. excentricus, which are known to produce highly potent neurotoxins known as ciguatoxins and have been associated with ciguatera outbreaks. Duplex PCR amplification of genomic DNA from strains of these species utilizing species-specific ssDNA tailed primers and a common primer containing the binding sequence of scCro DNA binding protein resulted in the generation of hybrid ssDNA-dsDNA amplicons. These were captured on the dipsticks via hybridization with complementary probes and detected with a scCro/carbon nanoparticle (scCro/CNPs) conjugate. The two different test zones on the dipsticks allowed the discrimination of the two species and the assay exhibited high sensitivity, 6.3 pg/µL of genomic DNA from both G. australes and G. excentricus. The specificity of the approach was also demonstrated using genomic DNA from non-target Gambierdiscus species and other microalgae genera which did not produce any signals. The possibility to use cells directly for amplification instead of purified genomic DNA suggested the compatibility of the approach with field sample testing. Future work is required to further explore the potential use of the strategy for on-site analysis and its applicability to other toxic species.

Hybrid Antibody-Aptamer Assay for Detection of Tetrodotoxin in Pufferfish.

The marine toxin tetrodotoxin (TTX) poses a great risk to public health safety due to its severe paralytic effects after ingestion. Seafood poisoning caused by the consumption of contaminated marine species like pufferfish due to its expansion to nonendemic areas has increased the need for fast and reliable detection of the toxin to effectively implement prevention strategies. Liquid chromatography-mass spectrometry is considered the most accurate method, although competitive immunoassays have also been reported. In this work, we sought to develop an aptamer-based assay for the rapid, sensitive, and cost-effective detection of TTX in pufferfish. Using capture-SELEX combined with next-generation sequencing, aptamers were identified, and their binding properties were evaluated. Finally, a highly sensitive and user-friendly hybrid antibody-aptamer sandwich assay was developed with superior performance compared to several assays reported in the literature and commercial immunoassay kits. The assay was successfully applied to the quantification of TTX in pufferfish extracts, and the results obtained correlated very well with a competitive magnetic bead-based immunoassay performed in parallel for comparison. This is one of the very few works reported in the literature of such hybrid assays for small-molecule analytes whose compatibility with field samples is also demonstrated.

Cyclodextrin polymers as passive sampling materials for lipophilic marine toxins in Prorocentrum lima cultures and a Dinophysis sacculus bloom in the NW Mediterranean Sea.

Cyclodextrins, cyclic oligomers that form a conical structure with an internal cavity, are proposed as new and sustainable materials for passive sampling of lipophilic marine toxins. Two applicability scenarios have been tested. First, disks containing ß-cyclodextrin-hexamethylene diisocyanate (ß-CD-HDI) and ß-cyclodextrin-epichlorohydrin (ß-CD-EPI) polymers were immersed in Prorocentrum lima cultures for different days (2, 12 and 40). LC-MS/MS analysis showed capture of free okadaic acid (OA) and dinophysistoxin-1 (DTX1) by cyclodextrins at contents that increased with immersion time. Cyclodextrins resulted more efficient in capturing DTX1 than OA. In a second experiment, disks containing ß-CD-HDI, ß-CD-EPI, γ-CD-HDI and γ-CD-EPI were deployed in harbor waters of El Masnou (NW Mediterranean Sea) during a Dinophysis sacculus bloom in February 2020. Free OA and pectenotoxin-2 (PTX2) were captured by cyclodextrins. Toxin contents were higher at sampling points and sampling weeks with higher D. sacculus cell abundance. In this case, PTX2 capture with cyclodextrins was more efficient than OA capture. Therefore, cyclodextrins have provided information regarding the toxin profile of a P. lima strain and the spatial and temporal dynamics of a D. sacculus bloom, proven efficient as passive sampling materials for environmental monitoring.

Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring.

The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.

Further advance of Gambierdiscus Species in the Canary Islands, with the First Report of Gambierdiscus belizeanus.

Ciguatera Poisoning (CP) is a human food-borne poisoning that has been known since ancient times to be found mainly in tropical and subtropical areas, which occurs when fish or very rarely invertebrates contaminated with ciguatoxins (CTXs) are consumed. The genus of marine benthic dinoflagellates Gambierdiscus produces CTX precursors. The presence of Gambierdiscus species in a region is one indicator of CP risk. The Canary Islands (North Eastern Atlantic Ocean) is an area where CP cases have been reported since 2004. In the present study, samplings for Gambierdiscus cells were conducted in this area during 2016 and 2017. Gambierdiscus cells were isolated and identified as G. australes, G. excentricus, G. caribaeus, and G. belizeanus by molecular analysis. In this study, G. belizeanus is reported for the first time in the Canary Islands. Gambierdiscus isolates were cultured, and the CTX-like toxicity of forty-one strains was evaluated with the neuroblastoma cell-based assay (neuro-2a CBA). G. excentricus exhibited the highest CTX-like toxicity (9.5-2566.7 fg CTX1B equiv. cell-1) followed by G. australes (1.7-452.6.2 fg CTX1B equiv. cell-1). By contrast, the toxicity of G. belizeanus was low (5.6 fg CTX1B equiv. cell-1), and G. caribaeus did not exhibit CTX-like toxicity. In addition, for the G. belizeanus strain, the production of CTXs was evaluated with a colorimetric immunoassay and an electrochemical immunosensor resulting in G. belizeanus producing two types of CTX congeners (CTX1B and CTX3C series congeners) and can contribute to CP in the Canary Islands.

Gambierdiscus and Fukuyoa as potential indicators of ciguatera risk in the Balearic Islands.

Gambierdiscus and Fukuyoa are genera of toxic dinoflagellates which were mainly considered as endemic to marine intertropical areas, and that are well known as producers of ciguatoxins (CTXs) and maitotoxins (MTXs). Ciguatera poisoning (CP) is a human poisoning occurring after the consumption of fish or more rarely, shellfish containing CTXs. The presence of these microalgae in a coastal area is an indication of potential risk of CP. This study assesses the risk of CP in the Balearic Islands (Western Mediterranean Sea) according to the distribution of both microalgae genera, and the presence of CTX-like and MTX-like toxicity in microalgal cultures as determined by neuro-2a cell based-assay (neuro-2a CBA). Genetic identification of forty-three cultured microalgal strains isolated from 2016 to 2019 revealed that all of them belong to the species G. australes and F. paulensis. Both species were widely distributed in Formentera, Majorca and Minorca. Additionally, all strains of G. australes and two of F. paulensis exhibited signals of CTX-like toxicity ranging respectively between 1 and 380 and 8-16 fg CTX1B equivalents (equiv.) • cell-1. Four extracts of F. paulensis exhibited a novel toxicity response in neuro-2a cells consisting of the recovery of the cell viability in the presence of ouabain and veratridine. In addition, G. australes showed MTX-like toxicity while F. paulensis strains did not. Overall, the low CTX-like toxicities detected indicate that the potential risk of CP in the Balearic Islands is low, although, the presence of CTX-like and MTX-like toxicity in those strains reveal the necessity to monitor these genera in the Mediterranean Sea.

A Single-Tube HNB-Based Loop-Mediated Isothermal Amplification for the Robust Detection of the Ostreid herpesvirus 1.

The Ostreid herpesvirus 1 species affects shellfish, contributing significantly to high economic losses during production. To counteract the threat related to mortality, there is a need for the development of novel point-of-care testing (POCT) that can be implemented in aquaculture production to prevent disease outbreaks. In this study, a simple, rapid and specific colorimetric loop-mediated isothermal amplification (LAMP) assay has been developed for the detection of Ostreid herpesvirus1 (OsHV-1) and its variants infecting Crassostrea gigas (C. gigas). The LAMP assay has been optimized to use hydroxynaphthol blue (HNB) for visual colorimetric distinction of positive and negative templates. The effect of an additional Tte UvrD helicase enzyme used in the reaction was also evaluated with an improved reaction time of 10 min. Additionally, this study provides a robust workflow for optimization of primers for uncultured viruses using designed target plasmid when DNA availability is limited.

Detecting harmful algal blooms with nucleic acid amplification-based biotechnological tools.

Harmful algal blooms (HABs) represent a growing threat to aquatic ecosystems and humans. Effective HAB management and mitigation efforts strongly rely on the availability of timely and in-situ tools for the detection of microalgae. In this sense, nucleic acid-based (molecular) methods are being considered for the unequivocal identification of microalgae as an attractive alternative to the currently used time-consuming and laboratory-based light microscopy techniques. This review provides an overview of the progress made on new molecular biotechnological tools for microalgal detection, particularly focusing on those that combine a nucleic acid (DNA or RNA) amplification step with detection. Different types of amplification processes (thermal and isothermal) and detection formats (e.g. microarrays, biosensors, lateral flows) are presented, and a comprehensive overview of their advantages and limitations is provided Although isothermal techniques are an attractive alternative to thermal amplification to reach in-situ analysis, further development is still required. Finally, current challenges, critical steps and future directions of the whole analysis process (from sample procurement to in-situ implementation) are described.