Study of a toxin-alkaline phosphatase conjugate for the development of an immunosensor for tetrodotoxin determination

This paper describes a direct competitive immunoenzymatic spectrophotometric assay (ELISA) for tetrodotoxin (TTX) determination and the adaptation of this method for use in an electrochemical assay format. The novelty of this work involves the use of the antigen labelled with alkaline phosphatase (AP); this conjugate was prepared in our laboratory as there is no commercially available conjugate of any kind for TTX. The new conjugate was characterized in terms of its affinity for the specific antibody as well as the residual concentration and the residual activity of the enzyme (AP) incorporated as label. The proposed method based on the new conjugate showed satisfactory results for TTX determination: for the spectrophotometric method the dynamic range was 4-15 ng mL(-1) with a limit of detection (LOD) of 2 ng mL(-1) (R=0.9247), whereas for the electrochemical protocol the dynamic range was 2-50 ng mL(-1) and the LOD was 1 ng mL(-1).