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.

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.

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.

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.

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.

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.

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.

Addressing the Analytical Challenges for the Detection of Ciguatoxins Using an Electrochemical Biosensor.

The importance of ciguatoxins (CTXs) in seafood safety and their emerging occurrence in locations far away from tropical areas highlight the need for simple and low-cost methods for the sensitive and rapid detection of these potent marine toxins to protect seafood consumers. Herein, an electrochemical immunosensor for the detection of CTXs is presented. A sandwich configuration is proposed, using magnetic beads (MBs) as immobilization supports for two capture antibodies, with their combination facilitating the detection of CTX1B, CTX3C, 54-deoxyCTX1B, and 51-hydroxyCTX3C. PolyHRP-streptavidin is used for the detection of the biotinylated detector antibody. Experimental conditions are first optimized using colorimetry, and these conditions are subsequently used for electrochemical detection on electrode arrays. Limits of detection at the pg/mL level are achieved for CTX1B and 51-hydroxyCTX3C. The applicability of the immunosensor to the analysis of fish samples is demonstrated, attaining detection of CTX1B at contents as low as 0.01 µg/kg and providing results in correlation with those obtained using mouse bioassay (MBA) and cell-based assay (CBA), and confirmed by liquid chromatography coupled to high-resolution mass spectrometry (LC-ESI-HRMS). This user-friendly bioanalytical tool for the rapid detection of CTXs can be used to mitigate ciguatera risk and contribute to the protection of consumer health.

Detection of tetrodotoxins in juvenile pufferfish Lagocephalus sceleratus (Gmelin, 1789) from the North Aegean Sea (Greece) by an electrochemical magnetic bead-based immunosensing tool.

Two small Lagocephalus sceleratus juveniles were captured in picarel targeting catches from North Aegean Sea (Greece) in the autumn of 2017. An electrochemical immunosensing tool using magnetic beads as immobilisation support was developed and applied to the rapid screening of tetrodotoxins (TTXs), potent neurotoxins that constitute a food safety hazard when present in seafood. This tool revealed the presence of TTXs in both individuals. Results were compared with those provided by mELISA and LC-HRMS, the latter confirming the presence of TTX. Some of the tissues contained TTX contents close to or above 2 mg/kg. L. sceleratus juveniles had been considered as non-toxic and, to our knowledge, this is the first report of high TTX levels in small L. sceleratus individuals. Such specimens can be mistaken with other edible species, posing a threat to consumers. The availability of low-cost and user-friendly tools for TTXs detection will contribute to guarantee seafood safety.

Rapid screening and multi-toxin profile confirmation of tetrodotoxins and analogues in human body fluids derived from a puffer fish poisoning incident in New Caledonia.

In August 2014, a puffer fish poisoning incidence resulting in one fatality was reported in New Caledonia. Although tetrodotoxin (TTX) intoxication was established from the patients' signs and symptoms, the determination of TTX in the patient's urine, serum or plasma is essential to confirm the clinical diagnosis. To provide a simple cost-effective rapid screening tool for clinical analysis, a maleimide-based enzyme-linked immunosorbent assay (mELISA) adapted for the determination of TTX contents in human body fluids was assessed. The mELISA was applied to the analysis of urine samples from two patients and a response for the presence of TTX and/or structurally similar analogues was detected in all samples. The analysis by LC-MS/MS confirmed the presence of TTX but also TTX analogues (4-epiTTX, 4,9-anhydroTTX and 5,6,11-trideoxyTTX) in the urine. A change in the multi-toxin profile in the urine based on time following consumption was observed. LC-MS/MS analysis of serum and plasma samples also revealed the presence of TTX (32.9 ng/mL) and 5,6,11-trideoxyTTX (374.6 ng/mL) in the post-mortem plasma. The results provide for the first time the TTX multi-toxin profile of human samples from a puffer fish intoxication and clearly demonstrate the implication of TTX as the causative agent of the reported intoxication case.

Development and validation of a maleimide-based enzyme-linked immunosorbent assay for the detection of tetrodotoxin in oysters and mussels.

The recent detection of tetrodotoxins (TTXs) in puffer fish and shellfish in Europe highlights the necessity to monitor the levels of TTXs in seafood by rapid, specific, sensitive and reliable methods in order to protect human consumers. A previous immunoassay for TTX detection in puffer fish, based on the use of self-assembled monolayers (SAMs) for the immobilization of TTX on maleimide plates (mELISA), has been modified and adapted to the analysis of oyster and mussel samples. Changing dithiol for cysteamine-based SAMs enabled reductions in the assay time and cost, while maintaining the sensitivity of the assay. The mELISA showed high selectivity for TTX since the antibody did not cross-react with co-occurring paralytic shellfish poisoning (PSP) toxins and no interferences were observed from arginine (Arg). Moreover, TTX-coated maleimide plates stored for 3 months at -20°C and 4°C were stable, thus when pre-prepared, the time to perform the assay is reduced. When analyzing shellfish samples, matrix effects and toxin recovery values strongly depended on the shellfish type and the sample treatment. Blank oyster extracts could be directly analyzed without solid-phase extraction (SPE) clean-up, whereas blank mussel extracts showed strong matrix effects and SPE and subsequent solvent evaporation were required for removal. However, the SPE clean-up and evaporation resulted in toxin loss. Toxin recovery values were taken as correction factors (CFs) and were applied to the quantification of TTX contents in the analysis of naturally-contaminated shellfish samples by mELISA. The lowest effective limits of detection (eLODs) were about 20 and 50µg/kg for oyster extracts without and with SPE clean-up, respectively, and about 30µg/kg for mussel extracts with both protocols, all of them substantially below the eLOD attained in the previous mELISA for puffer fish (230µg/kg). Analysis of naturally-contaminated samples by mELISA and comparison with LC-MS/MS quantifications demonstrated the viability of the approach. This mELISA is a selective and sensitive tool for the rapid detection of TTX in oyster and mussel samples showing promise to be implemented in routine monitoring programs to protect human health.

Self-assembled monolayer-based immunoassays for okadaic acid detection in seawater as monitoring tools.

Rapid and cost-effective methods to monitor the presence of diarrhetic shellfish poisoning (DSP) toxins in seawater samples in an easy and reliable manner are required to protect human health and avoid economic losses to shellfish industry. Immunoassays for the detection of okadaic acid (OA) and dinophysistoxin-1 and dinophysistoxin-2 are developed by immobilising OA on self-assembled monothiols or dithiols in an ordered and oriented way, providing an effective limit of detection of ∼1 ng OA equiv./mL seawater. The immunoassays are applied to the analysis of the particulate fraction of seawater samples from two Catalan harbours (NW Mediterranean) and samples collected periodically from the Galician Rias (E Atlantic), as well as a reference mussel sample. Results are in agreement with LC-MS/MS and the certified values. OA concentration in seawater correlates with Dinophysis cell abundance, with a 1-2 weeks lag. The immunoassays provide powerful high-throughput analytical methods potentially applicable as alternative monitoring tools.

Immunorecognition magnetic supports for the development of an electrochemical immunoassay for azaspiracid detection in mussels.

As azaspiracids (AZAs) are being reported from the coastal waters of an increasing number of countries on a global scale, the need for rapid, simple and cost-effective methods to detect these marine toxins and protect seafood consumers' health is becoming evident. A magnetic bead (MB)-based direct immunoassay for the detection of AZAs, using protein G-coated MBs as supports for antibody immobilisation and peroxidase-labelled AZA as a tracer is detailed. A colorimetric approach was first developed to optimise the experimental parameters and establish the cross-reactivity factors for AZA-1-10. The subsequent combination of the immunorecognition MBs with 8-electrode arrays enabled the multiplexed electrochemical detection of AZAs. Naturally-contaminated mussel samples were analysed and the results obtained showed an excellent correlation with LC-MS/MS analysis. The MB-based immunoassay facilitated the quantification of a wide range of AZA concentrations (120-2875µg AZA-1 equiv./kg), with a limit of detection (63µg AZA-1 equiv./kg) below the European regulatory threshold, using a protocol that requires very few steps and a short analysis time (~ 15min). The simplicity, cost-effectiveness, rapidity, robustness, selectivity and precision of the assay provide a valuable tool for the detection of all regulated AZAs and other toxic AZA analogues, suitable for end users in the field of food safety.