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Precolumn derivatization and high-efficiency liquid-phase fluorescence detection method for tetrodotoxin and kit based on the method

A technology of tetrodotoxin and pre-column derivatization, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of high detection cost and easy false positives in ELISA, and achieve low detection cost, high sensitivity, and avoid reagent errors.

Active Publication Date: 2014-08-13
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows us to detect certain chemical compounds called tetraodontoxynosum through their ability to change color when exposed to light or another stimulus. By doing this we have developed an assay system where there was only one type of molecular species involved but multiple types were possible due to its unique properties. These techniques could make our analysis faster and more accurate compared to previous methods like enzyme catalysis.

Problems solved by technology

The technical problem addressed in this patented text relating to the use of drugs called tetraodontotrichloroethane or tricincholinum ethylene dicholate (TETOCHLOR®)) during treatments on seafoods caused by harmful chemical substances like triphenol A (TrPA). This can lead to serious health issues if taken carelessly without being able to identify any specific contaminants that may have been present beforehand. Current testing techniques involve extracting samples containing these materials through various means followed by analysis based upon their properties. However, there exist concerns over potential safety hazards associated with animal sampling procedures. Additionally, current tests require complicated equipment and complex analytic processes which make it challenges to develop effective fluorochemistry assays suitable for identifying small amounts of drug residue left behind after treatment.

Method used

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  • Precolumn derivatization and high-efficiency liquid-phase fluorescence detection method for tetrodotoxin and kit based on the method
  • Precolumn derivatization and high-efficiency liquid-phase fluorescence detection method for tetrodotoxin and kit based on the method

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1 The detection of tetrodotoxin in the sample to be tested combined with C18 solid phase extraction column and WCX solid phase extraction column purification

[0041] (1) Extraction and purification of the sample: Weigh 2 g of the homogenized puffer fish sample, add 10 mL of 0.1% acetic acid, ultrasonically extract in a water bath at 85°C for 10 min, then boil in a water bath for 5 min, and centrifuge at 4500 r / min after cooling For 5 min, take all the supernatant and pass it through a C18 solid-phase extraction column (activated with 3mL methanol and 3mL 0.1% acetic acid before use), and then wash with 2mL 0.1% acetic acid; collect all the filtrate and adjust to pH with ammonia water =7.0, all of them passed through a WCX solid-phase extraction column (activated with 3mL of 10% ammonia methanol and 1mL of water before use), washed with 2mL of water, drained the column, and eluted with 5mL of 2% acetic acid-methanol. The eluate was blown dry with nitrogen in a...

Embodiment 2

[0045] Example 2 The detection of tetrodotoxin in the sample to be tested combined with tetrodotoxin affinity chromatography column purification

[0046] (1) Extraction and purification of the sample: Weigh 2 g of the homogenized puffer fish viscera sample, add 10 mL of 0.1% acetic acid, ultrasonically extract in a water bath at 85°C for 10 min, then boil in a water bath for 5 min, and centrifuge at 4500 r / min for 5 min after cooling. min, take all the supernatant, adjust the pH to 7.4 with 0.5mol / L disodium hydrogen phosphate solution, pass through the tetrodotoxin affinity chromatography column (3mL specification) at a speed of 2mL / min, and wash with 3mL PBS (0.01mol / L, pH=7.4), washed with 3mL deionized water, eluted with 5mL 1% acetic acid, and drained the column. The eluate was blown dry with nitrogen in a water bath at 90°C and dissolved in 1 mL of water to obtain a sample solution.

[0047] (2) Fluorescence derivatization: Take 0.4mL of the sample solution, add 0.2m...

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Abstract

The invention discloses a precolumn derivatization and high-efficiency liquid-phase fluorescence detection method for tetrodotoxin. The method includes following steps: (1) preparing a sample solution of a sample to be measured and a standard substance solution of tetrodotoaxin; (2) adding 0.2 ml of a pH maintenance solution to 0.4 ml of the sample solution for controlling pH of the sample solution at a range between 11.6 and 11.9, then adding 0.1 ml of a derivatization agent 1 and 0.1 ml of a derivatization agent 2, mixing uniformly, carrying out a water-bath heating process for 20 min and then carrying out a cooling process to room temperature, adding 0.1 ml of a stopping solution for enabling the pH of the sample solution to be neutral, and then carrying out a then diluting to volume with ultrapure water, adding the standard substance solution of the tetrodotoxin to the sample solution, mixing uniformly, and then carrying out a fluorescence derivatization process to the sample solution; and (3) with a liquid-phase chromatograph instrument equipped with a fluorescence detector, analyzing the sample solution which is subjected to the fluorescence derivatization process in the step (2) and the standard substance solution of the tetrodotoxin to detect the tetrodotoxin qualitatively and quantitatively in the sample solution. The invention also discloses a kit which is suitable for the method. The method is used for detecting the tetrodotoxin in aquatic products qualitatively and quantitatively and is reliable in result and low in detection limit.

Description

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Claims

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Application Information

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Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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