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Magnetic-nanoparticle-modification-based enzyme sensor used for detecting okadaic acid and preparation method of sensor

A technology of magnetic nanoparticles and okadaic acid, which is applied in the field of analysis and detection, can solve the problems of inability to realize online, real-time rapid detection, and low stability of PP2A enzyme, and achieve intuitive detection results, high detection structure accuracy, and short detection cycle short 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

Okadaic acid is a potent inhibitor of protein phosphatase 2A (Protein phosphatase2A, PP2A), which can inhibit the activity of protein phosphatase and lead to protein hyperphosphorylation. Using the property of okadaic acid to inhibit the activity of PP2A enzyme, it has been developed and established the protein phosphatase activity inhibition method of okadaic acid, however, the stability of non-immobilized PP2A enzyme is not high, and online, real-time rapid detection cannot be realized

Method used

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  • Magnetic-nanoparticle-modification-based enzyme sensor used for detecting okadaic acid and preparation method of sensor
  • Magnetic-nanoparticle-modification-based enzyme sensor used for detecting okadaic acid and preparation method of sensor
  • Magnetic-nanoparticle-modification-based enzyme sensor used for detecting okadaic acid and preparation method of sensor

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Experimental program
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Effect test

Embodiment 1

[0034] Add 30ul of Ni-IDA magnetic nanoparticle colloidal suspension with a diameter of 300nm into a microtube pre-filled with 300ul of binding buffer, mix well, and place the microtube on a magnetic stand (Adem Mag SV magnetic support, France ) to remove the liquid in the microtube, thus completing the cleaning of the magnetic nanoparticles. Repeat the cleaning once according to the above steps to fully remove non-specific binding, wherein the binding buffer is 20mM Tris solution with pH 7.5, solution Contains 500mM NaCl and 0.09% sodium azide.

[0035] After the Ni-IDA magnetic nanoparticles are fully washed, add 0.5ml of 1 unit / ul PP2A enzyme solution into the microtube, and shake the microtube at a speed of 800 rpm for 15min to make the magnetic nanoparticle colloidal suspension and the gene The engineered PP2A enzyme is fully coupled and adsorbed. After the reaction, the microtube is placed on the magnetic stand to remove the solution, and the magnetic nanoparticle-modifi...

Embodiment 2

[0038] Add 15ul of Ni-IDA magnetic nanoparticle colloidal suspension with a diameter of 300nm into a microtube pre-filled with 150ul of binding buffer, mix well, place the microtube on a magnetic stand to remove the liquid in the microtube, In this way, the cleaning of the magnetic nanoparticles is completed. Repeat the cleaning once according to the above steps to fully remove non-specific binding, wherein the binding buffer is 20mM Tris solution with pH 7.5, and the solution contains 500mM NaCl and 0.09% sodium azide.

[0039] After the Ni-IDA magnetic nanoparticles are fully washed, add 0.45ml of 1 unit / ul PP2A enzyme solution into the microtube, and shake the microtube at a speed of 750 rpm for 18min to make the magnetic nanoparticle colloidal suspension and the gene The engineered PP2A enzyme is fully coupled and adsorbed. After the reaction, the microtube is placed on the magnetic stand to remove the solution, and the magnetic nanoparticle-modified PP2A enzyme is obtained...

Embodiment 3

[0042] Add 20ul of Ni-IDA magnetic nanoparticle colloidal suspension with a diameter of 350nm into a microtube pre-filled with 200ul of binding buffer, mix well, place the microtube on a magnetic stand to remove the liquid in the microtube, In this way, the cleaning of the magnetic nanoparticles is completed. Repeat the cleaning once according to the above steps to fully remove non-specific binding, wherein the binding buffer is 20mM Tris solution with pH 7.5, and the solution contains 500mM NaCl and 0.09% sodium azide.

[0043] After the Ni-IDA magnetic nanoparticles are fully washed, add 0.6ml of 2 units / ul PP2A enzyme solution into the microtube, and shake the microtube at 850 rpm for 20 minutes to make the magnetic nanoparticle colloidal suspension and the gene The engineered PP2A enzyme is fully coupled and adsorbed. After the reaction, the microtube is placed on the magnetic stand to remove the solution, and the magnetic nanoparticle-modified PP2A enzyme is obtained. Was...

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Abstract

The invention relates to a magnetic-nanoparticle-modification-based enzyme sensor used for detecting okadaic acid. The enzyme sensor comprises an insulated base body, and an electrode system including a work electrode, a reference electrode and a counter electrode, which is formed on the base body, wherein a biological enzyme layer is fixed on the work electrode, and comprises magnetic nanoparticle modified protein phosphatase 2A (PP2A) and an electronic transfer body, the diameters of magnetic nanoparticles are 250nm-350nm, and the invention also relates to a preparation method of the enzyme sensor. Compared with a common enzyme sensor, the enzyme sensor has the advantages that the loading rate of PP2A on the surface of the work electrode is improved through the magnetic nanoparticle modified PP2A, so that the current response value of the enzyme sensor is improved by 10 times or more compared with the common PP2A sensor, and when the enzyme sensor is used for detecting okadaic acid of an aquatic product, the detection limit is lower than 0.20microgram / L, the reproducibility RSD is smaller than 5%, and the detection time consumption is smaller than 0.5h.

Description

technical field [0001] The invention relates to the technical field of analysis and detection, in particular to an enzyme sensor based on magnetic nanoparticle modification and a preparation method for detecting the shellfish toxin okadaic acid. 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 e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327G01N27/26
Inventor 袁高峰孙海燕方旭波金标旺
Owner ZHEJIANG OCEAN UNIV
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