Integrating new immunoassays, biosensors and cytotoxicity assays in the detection and quantification of the emerging marine toxins tetrodotoxins and ciguatoxins

Abstract

Considering the food safety risk emerging marine toxins such as tetrodotoxins (TTXs) and ciguatoxins (CTXs) present in seafood may pose to consumers, there is a real need to develop new methods capable of detecting these toxins in food, and thus, ensure consumers protection. To achieve this goal, this thesis has been mainly focused on the development of immunoassays, cytotoxic assays and immunosensors for the detection of emerging marine toxins implicated in seafood safety: TTXs and CTXs. The rapid, cost-effective, sensitive and reliable bioanalytical tools described herein are promising early warning tools alternative to the reference methods for the screening and quantification of TTXs and CTXs in fish, shellfish and microalgae. Chapter 1 consists of a general introduction about the current state of art of the emerging marine toxins TTXs and CTXs. This chapter describes the problematic issues derived from the presence of these toxins in seafood products for human health. It also describes how relevant is the development of methods for their detection. Chapter 2 includes the general and specific objectives of the thesis. Chapter 3 comprises two parts. The first one gives a detailed overview of the chemical properties, mechanisms of action, human health implications of several emerging marine toxins, and the different alternative biochemical methods for their detection. In the second part, the advances that offer the integration of bionanotechnology materials in the development of electrochemical immunosensors for the detection of toxins are described. Chapter 4 includes 5 scientific contributions. In the first work, an enzyme-linked immunosorbent assay based on the use of self-assembled monolayers for the TTX immobilisation on maleimide plates (mELISA) is developed. The applicability of the mELISA to the analysis of puffer fish samples is evaluated. In the second work, the mELISA is modified in order to decreased the assay in time and cost and adapted to enable the quantification of TTXs in oysters and mussels. In the third work, the SAM-based immobilisation approach employed in the mELISA is used for the development of an electrochemical immunosensor, and the applicability of the later to the analysis of puffer fish is investigated. The fourth work describes the development of an optical planar waveguide immunosensor based on the immobilisation of TTX on nanoarrayed scaffolds. The applicability of the biosensor to the analysis of puffer fish is also studied. Finally, the fifth contribution shows the usefulness of combining the mELISA as screening tool with the LC-MS/MS analysis as confirmatory technique for the quantification of TTXs contents in puffer fish from the Western Mediterranean coast. Chapter 5 includes two scientific contributions. In the first one, a multi-disciplinary approach combining the use of cell-based assays (CBA), the mouse bioassay (MBA) and high-resolution mass spectrometry (HRMS) is described for the identification and quantification of CTXs in a shark implicated in a fatal food poisoning event in Madagascar. The second work demonstrates the suitability of the Neuro-2a CBA to identify and quantify the production of CTXs and maitotoxins (MTXs) in Gambierdiscus australes extracts from the Macaronesian Islands. Finally, Chapter 6 summarises the general conclusions and future work of the thesis.

Date of Award	28 Sept 2017
Original language	English
Supervisor	Mònica Campàs (Director), Jorge Diogène (Director) & Francisco Valero Barranco (Tutor)