Development of a rapid test for tetrodotoxin detection in seafood

Introduction: Intoxication by marine toxin tetrodotoxin (TTX) through the consumption of contaminated seafood has been consistently reported in Asia, and its recent emergence in Mediterranean Sea waters has been alarming. TTX impairs nervous signals, with the lethal dose for humans being 1-2 mg, and no antidote is known. A simple test for the rapid, cost-effective, and simple analysis of potentially contaminated food is thus required. The accurate detection of TTX relies on costly and time-consuming techniques due to its small size and the potential interferences from seafood components. Biosensors exploiting single-stranded DNA aptamers as bioreceptors have garnered increasing interest recently due to their high affinity and specificity, especially toward small-molecule targets. Our group previously reported the selection of novel TTX-binding aptamers for use in a hybrid sandwich assay in combination with an antibody. Herein, we report the development of a rapid lateral flow device (LFD) for the rapid and facile detection of TTX.

Methods: A TTX aptamer bioreceptor was immobilized on a nitrocellulose membrane, and an anti-TTX IgG antibody-gold nanoparticle conjugate was prepared for detection. Several parameters were optimized for enhanced sensitivity, and the device's stability was evaluated via an accelerated stability study. Finally, the device was used to detect TTX in extracts from contaminated pufferfish.

Results: The developed LFD successfully detected TTX in buffer and in contaminated pufferfish extracts in less than 30 min, exhibiting a limit of detection of ~0.3 ng/mL, suitable for TTX detection in seafood below the 2 mg TTX equivalents/kg limit established in Japan. No interferences were observed from other marine toxins, whereas the stability of the device was more than 1 year when stored at 4°C.

Conclusions: The LFD based on an aptamer–antibody hybrid sandwich assay exhibited high sensitivity, specificity and stability, and its application in the rapid analysis of contaminated seafood was demonstrated.