Laia Reverté

New advances in electrochemical biosensors for the detection of toxins: Nanomaterials, magnetic beads and microfluidics systems. A review

The use of nanotechnology in bioanalytical devices has special advantages in the detection of toxins of interest in food safety and environmental applications. The low levels to be detected and the small size of toxins justify the increasing number of publications dealing with electrochemical biosensors, due to their high sensitivity and design versatility. The incorporation of nanomaterials in their development has been exploited to further increase their sensitivity, providing simple and fast devices, with multiplexed capabilities. This paper gives an overview of the electrochemical biosensors that have incorporated carbon and metal nanomaterials in their configurations for the detection of toxins. Biosensing systems based on magnetic beads or integrated into microfluidics systems have also been considered because of their contribution to the development of compact analytical devices. The roles of these materials, the methods used for their incorporation in the biosensor configurations as well as the advantages they provide to the analyses are summarised.

Alternative methods for the detection of emerging marine toxins: biosensors, biochemical assays and cell-based assays.

The emergence of marine toxins in water and seafood may have a considerable impact on public health. Although the tendency in Europe is to consolidate, when possible, official reference methods based on instrumental analysis, the development of alternative or complementary methods providing functional or toxicological information may provide advantages in terms of risk identification, but also low cost, simplicity, ease of use and high-throughput analysis. This article gives an overview of the immunoassays, cell-based assays, receptor-binding assays and biosensors that have been developed for the screening and quantification of emerging marine toxins: palytoxins, ciguatoxins, cyclic imines and tetrodotoxins. Their advantages and limitations are discussed, as well as their possible integration in research and monitoring programs.

Detection of Tetrodotoxins in Puffer Fish by a Self-Assembled Monolayer-Based Immunoassay and Comparison with Surface Plasmon Resonance, LC-MS/MS, and Mouse Bioassay.

The increasing occurrence of puffer fish containing tetrodotoxin (TTX) in the Mediterranean could represent a major food safety risk for European consumers and threaten the fishing industry. The work presented herein describes the development of a new enzyme linked immunosorbent assay (mELISA) based on the immobilization of TTX through dithiol monolayers self-assembled on maleimide plates, which provides an ordered and oriented antigen immobilization and favors the antigen-antibody affinity interaction. The mELISA was found to have a limit of detection (LOD) of TTX of 0.23 mg/kg of puffer fish matrix. The mELISA and a surface plasmon resonance (SPR) immunosensor previously developed were employed to establish the cross-reactivity factors (CRFs) of 5,6,11-trideoxy-TTX, 5,11-deoxy-TTX, 11-nor-TTX-6-ol, and 5,6,11-trideoxy-4-anhydro-TTX, as well as to determine TTX equivalent contents in puffer fish samples. Results obtained by both immunochemical tools were correlated (R(2) = 0.977). The puffer fish samples were also analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding CRFs were applied to the individual TTX contents. Results provided by the immunochemical tools, when compared with those obtained by LC-MS/MS, showed a good degree of correlation (R(2) = 0.991 and 0.979 for mELISA and SPR, respectively). The mouse bioassay (MBA) slightly overestimated the CRF adjusted TTX content of samples when compared with the data obtained from the other techniques. The mELISA has been demonstrated to be fit for the purpose for screening samples in monitoring programs and in research activities.

Evaluation of tetrodotoxins in puffer fish caught along the Mediterranean coast of Spain. Toxin profile of Lagocephalus sceleratus

Abstract Although consumption of Tetraodontidae species is prohibited in the EU, intoxications are still reported. The evaluation of tetrodotoxins (TTXs) by mass spectrometry (LC‐MS/MS and LC‐HRMS) and a screening immunoassay (mELISA) in tetraodontid fishes caught along the Western Mediterranean Sea revealed high concentrations of TTXs in Lagocephalus sceleratus while no TTXs were identified in L. lagocephalus and Sphoeroides pachygaster individuals. The high TTXs content found in the L. sceleratus analysed herein demonstrate the occurrence of highly toxic puffer fish in the Western Mediterranean Sea. Being L. sceleratus a recent invasive species in the Mediterranean, surveillance, risk assessment and risk management measures are necessary. The strategy used within this research work could be a valuable tool for future food safety monitoring. Graphical abstract Presence of tetrodotoxins (TTXs) in different puffer fish species caught off the Spanish coast (Western Mediterranean Sea) was evaluated by liquid chromatography coupled to mass spectrometry (LC‐MS/MS). The only toxic species was Lagocephalus sceleratus, a recently invasive species in the Mediterranean. The toxin profile was confirmed by high resolution mass spectrometry (LC‐HRMS). An immunoassay (mELISA) also showed TTX presence, providing complementary functional information. Figure. No Caption available. HighlightsThis is the first toxicity assessment of the Lessepsian migrant pufferfish Lagocephalus sceleratus from the Spanish coast.This is the first report dealing with quantification of 11 tetrodotoxin analogues in various organs of L. sceleratus.Excellent correlation of LC‐MS/MS, LC‐HRMS and mELISA is obtained in the analysis of puffer fish samples.A user‐friendly screening and quantification analysis tool for tetrodotoxin is provided.

Identification of ciguatoxins in a shark involved in a fatal food poisoning in the Indian Ocean

Severe food poisoning events after the consumption of sharks have been reported since the 1940s; however, there has been no clear understanding of their cause. Herein, we report for the first time the presence of ciguatoxins (CTXs) in sharks. The identification by mass spectrometry of CTXs, including two new analogues, in a bull shark (Carcharhinus leucas) that was consumed by humans, causing the poisoning and death of 11 people in Madagascar in 2013 is described. Typical neurotoxic ciguatera symptoms were recorded in patients, and toxicological assays on extracts of the shark demonstrated CTX-like activity. These results confirm this episode as a ciguatera poisoning event and expand the range of pelagic fish species that are involved in ciguatera in the Indian Ocean. Additionally, gambieric acid D, a molecule originally described in CTX-producing microalgae, was identified for the first time in fish. This finding can contribute to a better understanding of trophic relations within food webs. The present work confirms that consumption of sharks from the Indian Ocean should be considered a ciguatera risk, and actions should be taken to evaluate its magnitude and risk in order to manage shark fisheries.

Biosensors for the Detection of Emerging Marine Toxins

Emerging marine toxins present in the environment are relevant for food safety issues. Researchers are currently putting special emphasis on the development of biosensors for their detection. Due to their structural complexity and the difficulty to produce the corresponding biorecognition molecules, the development of assays and biosensors for their detection has become a challenge. Compared to traditional detection techniques, biosensors can provide advantages in terms of sensitivity, specificity, design versatility, portability and multiplexed configurations. This chapter provides a critical overview of the immunosensors, receptor-based biosensors, cell-based biosensors and aptasensors that have been developed for the detection of palytoxins (PlTXs), brevetoxins (PbTXs) and tetrodotoxins (TTXs). Although only few biosensors for these emerging marine toxins have been described to date, the chapter reflects the promising advances made in this field.

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.