Gold nanoparticle probe-based immunoassay as a new tool for tetrodotoxin detection in puffer fish tissues

A competitive format immunoassay based on a gold nanoparticle-labelled monoclonal antibody (Mab) for the rapid detection of tetrodotoxin (TTX) in puffer fish tissues was developed. In the assay, two antibodies were employed. The first antibody supplied in sample pad was as detector reagent and the secondary antibody immobilized on a diagnostic cellulose nitrate membrane was as capture reagent. TTX in tissue sample competed with immobilized TTX–BSA to bind with gold nanoparticle-labelled Mab. The mobile complex (gold nanoparticle–Mab–TTX) can be captured by the secondary antibody but cannot be captured by TTX–BSA (test line). The color density of the test line correlated with the concentration of TTX in sample in the range 40–8000 ng mL−1. The visual detection limit of TTX-spiked samples was found to be 40 ng mL−1. The assay time of the qualitative test based on the visual evaluation of results for TTX detection was less than 10 min, and the assay performance did not require any equipment. This method can be applied as a screening method for rapid testing of TTX on site.