Biotechnological tools for the detection of tetrodotoxins in puffer fish and shellfish

The expanding occurrence of puffer fish and shellfish containing tetrodotoxins (TTXs) in Europe highlights the need to ensure seafood safety and to protect human health. Thus, specific, sensitive, fast, reliable,... [ view full abstract ]

The expanding occurrence of puffer fish and shellfish containing tetrodotoxins (TTXs) in Europe highlights the need to ensure seafood safety and to protect human health. Thus, specific, sensitive, fast, reliable, cost-effective and user-friendly detection methods for TTXs are required. With this aim, colorimetric immunoassays and electrochemical immunosensors were developed. Self-assembled monolayers (SAMs) were used for TTX immobilisation in order to favour antigen/antibody interaction in the competition assay and, in the electrochemical biosensors, to promote electron transfer. Colorimetric immunoassays (named mELISA because of the use of maleimide-activated plates for the monolayer assembly) were performed with TTX as well as with some TTX analogues. For these TTX analogues, cross-reactivity factors were established. Colorimetric mELISAs were applied to the detection of TTXs in puffer fish (several tissues) and shellfish (mussels and oysters). The limits of detection (LODs) were 0.23 mg equiv. TTX/kg puffer fish and 0.02 mg equiv. TTX/kg shellfish. Regarding the electrochemical immunosensor, 8 x gold electrode arrays were used and the electrochemical signal from appropriate redox mediators was recorded. The LOD attained was 0.07 mg equiv. TTX/kg puffer fish. Good agreements were found between the TTX quantifications obtained with the biotechnological tools and those provided by other techniques (surface Plasmon resonance, SPR, and liquid chromatography coupled to mass spectrometry, LC-MS). These biotechnological tools meet the requeriments in terms of LOD (the Japanese regulatory limit is 2 mg equiv. TTX/kg puffer fish), are suitable for screening purposes and also allow the accurate determination of TTX contents in fish and shellfish samples.