Automated Patch Clamp for the Detection of Tetrodotoxin in Pufferfish Samples

Abstract

Tetrodotoxin (TTX) is a marine toxin responsible for many intoxications around the world. Its presence in some pufferfish species and, as recently reported, in shellfish, poses a serious health concern. Although TTX is not routinely monitored, there is a need for fast, sensitive, reliable, and simple methods for its detection and quantification. In this work, we describe the use of an automated patch clamp (APC) system with Neuro-2a cells for the determination of TTX contents in pufferfish samples. The cells showed an IC50 of 6.4 nM for TTX and were not affected by the presence of muscle, skin, liver, and gonad tissues of a Sphoeroides pachygaster specimen (TTX-free) when analysed at 10 mg/mL. The LOD achieved with this technique was 0.05 mg TTX equiv./kg, which is far below the Japanese regulatory limit of 2 mg TTX equiv./kg. The APC system was applied to the analysis of extracts of a Lagocephalus sceleratus specimen, showing TTX contents that followed the trend of gonads > liver > skin > muscle. The APC system, providing an in vitro toxicological approach, offers the advantages of being sensitive, rapid, and reliable for the detection of TTX-like compounds in seafood.

Toxicity Equivalency Factors for Tetrodotoxin Analogues Determined with Automated Patch Clamp on Voltage-Gated Sodium Channels in Neuro-2a Cells

“…of 10 mg/mL. According to our previous works, potential coextracted compounds, if any, do not interfere with the TTX calibration curve at this matrix concentration and, therefore, there is no need to apply any recovery factor in the determination of TTX concentrations. The same applies to the determination of TTX concentrations by CBA, where the ouabain/veratridine controls indicated the absence of matrix effects at the tested matrix concentrations .…”

“…The electrophysiological changes of Neuro-2a cells in response to the TTX analogues were evaluated using a Patchliner (Nanion Technologies GmbH, Munich, Germany), an automated planar patch clamp device . Briefly, eight wells of a medium resistance NPC-16 borosilicate planar chip (Nanion Technologies GmbH, Munich, Germany) were filled up with the internal and external solutions.…”

“…The detection of toxins with CBA relies on assessing changes in the morphology or viability of cells after their exposure to them. − However, cells used for CBA sometimes require pretreatments with auxiliary drugs to ensure optimal assay performance, necessitating cautious interpretation of the toxicological data that they generate. Recently, a new approach that leverages the electrophysiological activity of Neuro-2a cells has emerged as a promising high throughput bioanalytical method for detecting TTXs in pufferfish samples, with potential applications in food safety and broader toxicological studies …”