Biosensor for detecting okadaic acid and preparation method

A kind of okadaic acid and biosensor technology, applied in the field of analysis and detection, can solve the problems of high cultivation period and cost, unintuitive detection results, unsuitable for rapid detection, etc., and achieve intuitive detection results, short detection cycle and rapid detection Effect

Inactive Publication Date: 2015-10-14
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the mouse biological method, this method has the advantages of conforming to the 3R principle, high sensitivity, good specificity, good repeatability, simple sample extraction, and low matrix effect; compared with the ELISA method, it has the advantages of better specificity and higher sensitivity , but in this method, cells are used as the experimental object, the culture cycle and cost are high, and the detection result is not intuitive, which is not suitable for the needs of rapid detection

Method used

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  • Biosensor for detecting okadaic acid and preparation method
  • Biosensor for detecting okadaic acid and preparation method
  • Biosensor for detecting okadaic acid and preparation method

Examples

Experimental program
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Effect test

Embodiment 1-1

[0028] Clean the PVC film substrate (Sigma-Aldrich, USA) with working electrode, reference electrode and counter electrode on the surface, and dry it for later use. Dissolve PP2A enzyme in pH 8.4 buffer A containing 30mM Tris–HCl, 2mM EDTA and 20mM MgCl2 to make 1 unit / ul enzyme solution, in which 1 unit of PP2A enzyme is cleaved within 1min at 25°C Amount of enzyme required for p-nitrophenyl phosphate disodium salt. In this enzyme solution, add PVA-AWP (polyvinyl alcohol-has (azide-unit pendant) water-soluble photopolymer of azide unit side group, Toyo Gosey Kogyo corporation, Japan), the quality of PP2A enzyme and PVA-AWP The ratio is 2:1 and a fixative solution is formed after homogenization. Then, 3 ul of fixing solution was added dropwise to the surface of the working electrode, and irradiated for 3 h at 4° C. under a fluorescent lamp with a power of 15 W. After the light was finished, it was further dried at 4°C for 24 hours, and then rinsed with double distilled water...

Embodiment 1-2

[0030]Clean the PVC film matrix with working electrode, reference electrode and counter electrode on the surface, and dry it for later use. Dissolve PP2A enzyme in pH 8 buffer A containing 30mM Tris-HCl, 2mM EDTA and 20mM MgCl2 to make 2 units / ul of enzyme solution, of which 1 unit of PP2A enzyme is cleaved within 1min at 25°C The amount of enzyme required for the disodium salt of p-nitrophenyl phosphate is described. Add PVA-AWP to the enzyme solution, the mass ratio of PP2A enzyme to PVA-AWP is 1:1, and form a fixed solution after homogenization. Then 3.5ul of fixative solution was added dropwise to the surface of the working electrode, and irradiated under a fluorescent lamp with a power of 15W for 4h at 5°C. After the light was finished, it was further dried at 3.5°C for 18 hours, and then rinsed with double distilled water to remove excess PP2A enzyme physically adsorbed. Finally, the prepared immobilized PP2A enzyme electrode was stored at 4°C for use.

Embodiment 1-3

[0032] Clean the PVC film matrix with working electrode, reference electrode and counter electrode on the surface, and dry it for later use. Dissolve PP2A enzyme in pH 8.7 buffer A containing 30mM Tris–HCl, 2mM EDTA and 20mM MgCl2 to make 1.5 units / ul enzyme solution, in which 1 unit of PP2A enzyme is cleaved within 1min at 25°C The amount of enzyme required for the disodium salt of p-nitrophenyl phosphate is described. Add PVA-AWP to the enzyme solution, the mass ratio of PP2A enzyme to PVA-AWP is 1.5:1, and form a fixed solution after homogenization. Then 3 ul of fixing solution was added dropwise to the surface of the working electrode, and irradiated under a fluorescent lamp with a power of 12W for 6 hours at 3°C. After the light was finished, it was further dried at 5°C for 12 hours, and rinsed with double distilled water after drying to remove excess PP2A enzyme physically adsorbed, and finally the prepared immobilized PP2A enzyme electrode was stored at 4°C for use.

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Abstract

The invention relates to a biosensor for detecting okadaic acid. The biosensor comprises an insulated substrate and an electrode system, wherein the electrode system is formed on the substrate and comprises a working electrode, a reference electrode and a counter electrode; and a biological enzyme layer is fixed on the working electrode and comprises protein phosphatase 2A (PP2A enzyme) and an electron transport complex. The invention further relates to a preparation method of the biosensor, and mainly relates to fixation of the PP2A enzyme. Compared with the prior art, the biosensor taking the PP2A enzyme as a fixed enzyme layer is prepared by utilizing the inhibition effect of okadaic acid to the activity of the PP2A enzyme; the content of okadaic acid in a sample is determined according to the activity inhibiting degree of the PP2A enzyme, namely, the concentration of okadaic acid in the sample is converted into an electric signal to be detected, and therefore, the detection result is visual; simultaneously, by virtue of the specificity and the high efficiency of PP2A enzyme catalytic reaction, the detection process is high in specificity, the accuracy rate of a detection structure is high, the detection period is short, and instant and rapid detection requirements can be met.

Description

technical field [0001] The invention relates to the technical field of analysis and detection, in particular to a biosensor for detecting the shellfish toxin okadaic acid and a preparation method thereof. Background technique [0002] Okadaic acid (OA), a small molecule marine polyether toxin, is one of the most widely distributed and most morbid marine toxins. It often accumulates in shellfish and other marine organisms, and can cause multiple A type of food poisoning characterized by diarrhea, which can lead to severe gastrointestinal dysfunction, and the symptoms of poisoning are easily confused with bacterial gastroenteritis. There is no specific medicine for okadaic acid poisoning, and in coastal areas, mussels contaminated by diarrheal shellfish toxins may be accidentally eaten, causing poisoning, which seriously affects people's health and the development and utilization of seafood. Therefore, it is particularly important to establish a rapid, sensitive and reliable ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26G01N27/327G01N27/30
Inventor 袁高峰孙海燕陈小娥唐文艳张晓娟
Owner ZHEJIANG OCEAN UNIV
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