TTX Detection Research Articles

Tetrodotoxin and the state-of-the-art progress of its associated analytical methods.

Tetrodotoxin (TTX), which is found in various marine organisms, including pufferfish, shellfish, shrimp, crab, marine gastropods, and gobies, is an effective marine toxin and the cause of many seafood poisoning incidents. Owing to its toxicity and threat to public health, the development of simple, rapid, and efficient analytical methods to detect TTX in various food matrices has garnered increasing interest worldwide. Herein, we reviewed the structure and properties, origin and sources, toxicity and poisoning, and relevant legislative measures of TTX. Additionally, we have mainly reviewed the state-of-the-art progress of analytical methods for TTX detection in the past five years, such as bioassays, immunoassays, instrumental analysis, and biosensors, and summarized their advantages and limitations. Furthermore, this review provides an in-depth discussion of the most advanced biosensors, including cell-based biosensors, immunosensors, and aptasensors. Overall, this study provides useful insights into the future development and wide application of biosensors for TTX detection.

Recent research progress in tetrodotoxin detection and quantitative analysis methods.

Tetrodotoxin (TTX) is a highly potent and widely distributed ion-channel marine neurotoxin; it has no specific antidote and poses a great risk to human health. Therefore, detecting and quantifying TTX to effectively implement prevention strategies is important for food safety. The development of novel and highly sensitive, highly specific, rapid, and simple techniques for trace TTX detection has attracted widespread attention. This review summarizes the latest advances in the detection and quantitative analysis of TTX, covering detection methods based on biological and cellular sensors, immunoassays and immunosensors, aptamers, and liquid chromatography-mass spectrometry. It further discusses the advantages and applications of various detection technologies developed for TTX and focuses on the frontier areas and development directions of TTX detection, providing relevant information for further investigations.

A three-channel biosensor based on stimuli-responsive catalytic activity of the Fe3O4@Cu for on-site detection of tetrodotoxin in fish

Tetrodotoxin (TTX), a lethal neurotoxin, poses a grave threat to human health. The available spectroscopic methods suffer from limitations such as complex procedures and inadequate on-site capabilities. In this study, we proposed a method using Fe3O4@Cu as a catalytic biosensor combined with SERS, colorimetry and image processing for TTX detection. Integrating the aptamer amplifies the specificity of the system and masks the catalytic activity of Fe3O4@Cu. The catalytic efficiency of Fe3O4@Cu in the H2O2-TMB reaction can quantify the concentration of TTX in the system. Consequently, oxidation of TMB (oxTMB) led to the generation and change of signals for SERS, colorimetry and image processing, enabling a three-channel quantitative detection of TTX. Under the optimal conditions, the detection limit of established SERS, colorimetry and image processing were 0.055, 2.127 and 0.243 ng/mL, respectively. This three-channel biosensor was applied to real samples, providing an accurate, stable and adaptable alternative for on-site TTX detection.

Tetrodotoxin and the Geographic Distribution of the Blue-Lined Octopus Hapalochlaena fasciata on the Korean Coast

The genus Hapalochlaena, including the blue-lined octopus Hapalochlaena fasciata (H. fasciata), is highly toxic. Venomous, blue-lined octopuses were recently found in Korea, but their toxicity, toxin composition, and distribution remain largely unknown. Here we estimated the geographic distribution of the organisms along the Korean coast and clarified their toxicity. Tetrodotoxin (TTX) was present in all three specimens of H. fasciata examined, although the toxicity varied largely between individuals. The mean TTX concentration in the whole body of the three specimens was 6.5 ± 2.2 μg/g (range 3.3-8.5 μg/g). Among the body parts examined, the salivary glands exhibited the highest concentration (22.4 ± 9.7 μg/g). From 2012 to 2021, 26 individuals were obtained nearly every month from different regions of the Korean coast. A non-fatal case of a blue-lined octopus bite was reported along the Korean coast in June 2015. This is the first report on the widespread distribution of blue-lined octopuses on the Korean coast and TTX detection. The widespread distribution of the TTX-bearing H. fasciata along the Korean coast within the temperate zone indicates that the species may soon become a serious health issue in Korea. The toxicity of this species is also a potentially significant human health risk.

An Overview of the Anatomical Distribution of Tetrodotoxin in Animals

Tetrodotoxin (TTX), a potent paralytic sodium channel blocker, is an intriguing marine toxin. Widely distributed in nature, TTX has attracted attention in various scientific fields, from biomedical studies to environmental safety concerns. Despite a long history of studies, many issues concerning the biosynthesis, origin, and spread of TTX in animals and ecosystems remain. This review aims to summarize the current knowledge on TTX circulation inside TTX-bearing animal bodies. We focus on the advances in TTX detection at the cellular and subcellular levels, providing an expanded picture of intra-organismal TTX migration mechanisms. We believe that this review will help address the gaps in the understanding of the biological function of TTX and facilitate the development of further studies involving TTX-bearing animals.

Facilely self-assembled and dual-molecule calibration aptasensor based on SERS for ultra-sensitive detection of tetrodotoxin in pufferfish

Tetrodotoxin (TTX) is one of the most lethal neurotoxins, so the reliable quantitative analysis of TTX is crucial for food and environmental safety monitoring. Herein, a novel dual-molecule calibration aptasensor was developed for detection of TTX based on Surface-enhanced Raman scattering (SERS). The adaptive surface has high affinity recognition sites for the target of interest, which ensures the high specificity and stability of the aptasensor. In addition, the uniquely labeled signal molecules located in the Raman silent region (1800–2400 cm−1) can avoid the interference of other exogenous biological signal molecules. Meanwhile, in quantitative analysis, the SERS signal generated by the reporter is calibrate in real time using the second-order peak of silicon molecule (Si). The detection linear range of the aptasensor was 0.0319 ng/mL-319.27 ng/mL, with a limit of detection (LOD) of 0.024 ng/mL and the excellent uniformity (RSD = 8.8%) for TTX detection. As a promising and versatile detection candidate, the ultra-sensitive quantitative detection aptasensor of TTX had important practical significance, which can offer more favorable persuasion for TTX analysis in real seafood samples.

Voltammetric Detection of Tetrodotoxin Real-Time In Vivo of Mouse Organs using DNA-Immobilized Carbon Nanotube Sensors

Background: Tetrodotoxin (TTX) is a biosynthesized neurotoxin that exhibits powerful anticancer and analgesic abilities by inhibiting voltage-gated sodium channels that are crucial for cancer metastasis and pain delivery. However, for the toxin’s future medical applications to come true, accurate, inexpensive, and real-time in vivo detection of TTX remains as a fundamental step. Methods: In this study, highly purified TTX extracted from organs of Takifugu rubripes was injected and detected in vivo of mouse organs (liver, heart, and intestines) using Cyclic Voltammetry (CV) and Square Wave Anodic Stripping Voltammetry (SWASV) for the first time. In vivo detection of TTX was performed with auxiliary, reference, and working herring sperm DNA-immobilized carbon nanotube sensor systems. Results: DNA-immobilization and optimization of amplitude (V), stripping time (sec), increment (mV), and frequency (Hz) parameters for utilized sensors amplified detected peak currents, while highly sensitive in vivo detection limits, 3.43 µg L-1 for CV and 1.21 µg L-1 for SWASV, were attained. Developed sensors herein were confirmed to be more sensitive and selective than conventional graphite rodelectrodes modified likewise. A linear relationship was observed between injected TTX concentration and anodic spike peak height. Microscopic examination displayed coagulation and abnormalities in mouse organs, confirming the powerful neurotoxicity of extracted TTX. Conclusion: These results established the diagnostic measures for TTX detection regarding in vivo application of neurotoxin-deviated anticancer agents and analgesics, as well as TTX from food poisoning and environmental contamination.

Detection of okadaic acid (OA) and tetrodotoxin (TTX) simultaneously in seafood samples using colloidal gold immunoassay

Tetrodotoxin (TTX) is a neurotoxin mainly responsible for severe neurological illness, and okadaic acid (OA) is another important lipophilic toxin to humans. In this study, we developed a gold strip for simultaneous detection of OA and TTX in real seafood samples. In the assay, the prepared nanoparticles (about 40 nm) was applied to conjugate with specific monoclonal antibodies against OA and TTX, and the resulted mixtures were used to capture its corresponding toxin in test strip. OA and TTX conjugates were coated as two test lines on the nitrocellulose membrane, and goat anti-mouse IgG was used to form the control line, forming three lines on the test strip. The visual detection limits (vLOD) of this immunoassay for OA and TTX were 0.75 and 15 ng/mL, respectively, and no cross reactions were observed in the process of detection. The visual assay for OA and TTX detection could be finished within 10 min. This study might provide a feasible method and good understanding for rapidly simultaneous detection for toxins based on immunoassay.

The Incidence of Tetrodotoxin and Its Analogs in the Indian Ocean and the Red Sea.

Tetrodotoxin (TTX) is a potent marine neurotoxin with bacterial origin. To date, around 28 analogs of TTX are known, but only 12 were detected in marine organisms, namely TTX, 11-oxoTTX, 11-deoxyTTX, 11-norTTX-6(R)-ol, 11-norTTX-6(S)-ol, 4-epiTTX, 4,9-anhydroTTX, 5,6,11-trideoxyTTX, 4-CysTTX, 5-deoxyTTX, 5,11-dideoxyTTX, and 6,11-dideoxyTTX. TTX and its derivatives are involved in many cases of seafood poisoning in many parts of the world due to their occurrence in different marine species of human consumption such as fish, gastropods, and bivalves. Currently, this neurotoxin group is not monitored in many parts of the world including in the Indian Ocean area, even with reported outbreaks of seafood poisoning involving puffer fish, which is one of the principal TTX vectors know since Egyptian times. Thus, the main objective of this review was to assess the incidence of TTXs in seafood and associated seafood poisonings in the Indian Ocean and the Red Sea. Most reported data in this geographical area are associated with seafood poisoning caused by different species of puffer fish through the recognition of TTX poisoning symptoms and not by TTX detection techniques. This scenario shows the need of data regarding TTX prevalence, geographical distribution, and its vectors in this area to better assess human health risk and build effective monitoring programs to protect the health of consumers in Indian Ocean area.

Tetrodotoxin-Producing Bacteria: Detection, Distribution and Migration of the Toxin in Aquatic Systems.

This review is devoted to the marine bacterial producers of tetrodotoxin (TTX), a potent non-protein neuroparalytic toxin. In addition to the issues of the ecology and distribution of TTX-producing bacteria, this review examines issues relating to toxin migration from bacteria to TTX-bearing animals. It is shown that the mechanism of TTX extraction from toxin-producing bacteria to the environment occur through cell death, passive/active toxin excretion, or spore germination of spore-forming bacteria. Data on TTX microdistribution in toxic organs of TTX-bearing animals indicate toxin migration from the digestive system to target organs through the transport system of the organism. The role of symbiotic microflora in animal toxicity is also discussed: despite low toxin production by bacterial strains in laboratory conditions, even minimal amounts of TTX produced by intestinal microflora of an animal can contribute to its toxicity. Special attention is paid to methods of TTX detection applicable to bacteria. Due to the complexity of toxin detection in TTX-producing bacteria, it is necessary to use several methods based on different methodological approaches. Issues crucial for further progress in detecting natural sources of TTX investigation are also considered.

Detection of tetrodotoxin-producing Providencia rettgeri T892 in Lagocephalus pufferfish

Tetrodotoxin (TTX) is a potent toxin but it could be used in pharmaceutical field. Identification of TTX producing bacteria in pufferfish is necessary for TTX yield and the pufferfish conservation. In this study, Lagocephalus was collected from Cam Ranh Sea, a central part of Vietnam during spring season. The liver and intestine were incubated in 0.9% NaCl for TTX detection in pufferfish. To be benefited from the isolation of new TTX producing bacteria, the liver and intestine were incubated in 6.5% NaCl. The cultures were used to test the toxin and to isolate the bacterial community that could yield TTX. Surprisingly, Providencia rettgeri T892 in intestine could produce TTX identified by biochemical test and 16S rRNA sequencing. This strain was used to test the production of TTX, based on thin layer chromatography (TLC), mouse bioassay and high performance liquid chromatography (HPLC) analysis. The bacterium was optimized for TTX production in media prepared from the meat-washing water of Auxis thazard, Megalaspis cordyla and Decapterus maruadsi. Interestingly, the TTX obtained 0.106mg/mL and 0.055mg/mL in medium prepared from A. thazard and M. cordyla, respectively while there was no TTX production detected in medium prepared from D. maruadsi. This paper could contribute to warn to the human health care system about a possible TTX poisoning in some cases related to eating fishes.

Development of Direct Competitive ELISA Kit for the Detection of Tetrodotoxin Using HRP Labeled Antigen

The direct competitive enzyme-linked immunosorbent assay (dcELISA) kit was developed for detecting tetrodotoxin (TTX). The working conditions of the dcELISA kit including the anti-TTX mAb coating concentration, coating method, enzyme-labeled antigen concentration, the antigen diluents, reaction time and temperature were all optimized. The result showed that mAb coating concentration was 3.72 μg/ml, it was coated at the condition of minimal power treatment of microwave oven for 3 min. The enzyme-labeled antigen concentration was 4.08 μg/ml. The competitive reaction was under the condition of room temperature 25 °C for 30 min. The half maximal inhibitory concentration (IC50) of the standard curve was 20.4 ng/ml, detection limit was 1.1 ng/ml, linear range 3.3～137 ng/ml, the intra-assay CV and inter-assay CV were 6.25% and 7.34% respectively. And recovery rate of TTX ranged from 65.0% to 93.2% with the CV of 9.41～12.77%. This method is convenient, sensitive and time-saving, hope this dcELISA kit can bring benefits and reference for TTX detection.

Evaluation of Surface Plasmon Resonance Biosensors for Detection of Tetrodotoxin in Food Matrices and Comparison to Analytical Methods

Tetrodotoxin (TTX) is a low molecular weight neurotoxin found in a number of animal species, including pufferfish. One emerging method for TTX detection employs surface plasmon resonance (SPR) immunosensors. SPR, an optical technique that allows for label-free, real-time, multiplexed analysis, can have detection limits that rival many of the conventional transduction methods. Preliminary SPR approaches for TTX were successful, yet suffered from low throughput and used noncommercial instrumentation. To advance this method for broader use, the immunoassay was transferred to a commercial instrument and optimized for improved detection. This manuscript outlines the assay development and results for complex matrices relevant to seafood safety (pufferfish) and food adulteration (milk, apple juice). In addition, results are compared to those obtained using receptor binding assay, ELISA, HPLC-FD, and LC/MS/MS detection techniques. Results highlight the advantages of SPR assays, including rapid screening capability with low reagent consumption and low- to subppb detection limits.

CURRENT METHODOLOGIES FOR DETECTION OF TETRODOTOXIN

In attempt to protect the consumers from TTX-intoxication, the mouse bioassay has historically been the most universally applied tool to determine the toxicity level in monitoring programs. This bioassay, however, shows low precision and requires a continuous supply of a particular size of mice. These potential drawbacks and the world-wide pressure of refraining from the killing of the live animals subsequently led the scientists for developing alternative chemical methods to the mouse bioassay for TTX detection and quantification. Among them, high-performance liquid chromatography (HPLC) has largely been used in routine work.