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ELISA test method on basis of nanometer enzyme with peroxidase activity

A technology of peroxidase and detection method, which is applied in the field of ELISA detection of nanozymes, can solve the problems of unstable catalytic activity and reduced catalytic activity of nanozymes, so as to avoid the decline of enzyme catalytic activity, strong catalytic activity and stable reproduction sexual effect

Active Publication Date: 2016-10-12
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art, providing a kind of ELISA detection method based on nanozyme, this kind of nanozyme is prepared by the method for silver-platinum staining, has peroxidase activity, the present invention can solve the present invention In the existing technology, the catalytic activity of nanozymes is unstable, and it is easy to reduce the catalytic activity due to modification

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  • ELISA test method on basis of nanometer enzyme with peroxidase activity
  • ELISA test method on basis of nanometer enzyme with peroxidase activity
  • ELISA test method on basis of nanometer enzyme with peroxidase activity

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1: Preparation of gold nanoparticles

[0043] Add 100mL 0.01wt% chloroauric acid into a round bottom flask, boil and reflux under continuous stirring, add 1mL 3wt% sodium citrate at a constant speed, continue stirring and boiling for 30min, and synthesize gold nanoparticles (AuNPs) with a particle size of 16nm. By adjusting the ratio of chloroauric acid and sodium citrate, gold nanoparticles with different particle sizes can be obtained.

Embodiment 2

[0044] Embodiment 2: the comparative experiment of silver platinum dyeing condition

[0045] In this example, bovine serum albumin (BSA) was used to modify gold nanoparticles. Of course, the gold nanoparticles obtained in Example 1 can also be directly stained with silver and platinum without BSA modification, and nanozymes with peroxidase activity can also be obtained.

[0046] First, 100 μL of 2wt% BSA / PBS solution was added to the 96-well plate, incubated at 37° C. for 1 h, and washed three times with 300 μL of PBS. Subsequently, 100 μL of 2.5 nM 16 nm AuNPs prepared in Example 1 (no AuNPs were added to the control group), incubated at 37° C. for 1 h, and washed three times with 300 μL of PBS. Then add 300 μL Blocking Buffer (PBS solution containing 2wt% BSA and 0.1wt% Tween20), incubate at 37°C for 1 h, and wash with 300 μL PBS three times to obtain BSA-modified gold nanoparticles Au / BSA, which are wrapped and adsorbed on the surface of a 96-well plate .

[0047] Subseq...

Embodiment 3

[0050] Example 3: Characterization of nanozyme Au / BSA@AgPt particles with peroxidase activity obtained after silver-platinum staining

[0051] Add AgNO to the BSA-modified gold nanoparticles Au / BSA obtained in 1mL 2.5nM Example 2 3 solution and hydroquinone and make Ag + and hydroquinone at a final concentration of 125 μM and 0.2 mM, respectively, and reacted at 37° C. for 20 minutes. Wash with 300 μL water three times to obtain Au / BSA@Ag particles. Then add H 2 PtCl 6 solution and ascorbic acid and make PtCl 6 2- The final concentrations of ascorbic acid and ascorbic acid were 0.5mM and 10mM, respectively, reacted at 37°C for 20min, and washed three times with 300μL water to obtain nanozyme Au / BSA@AgPt particles with peroxidase activity. The gold nanoparticles before and after silver staining were characterized by camera, ultraviolet-visible absorption spectrometer, scanning electron microscope and high-resolution transmission electron microscope.

[0052] The result i...

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Abstract

The invention discloses an ELISA test method on the basis of nanometer enzyme with peroxidase activity. The ELISA test method is fit for a double antibody sandwich method or a double antigen sandwich method. The ELISA test method comprises the following steps: modifying gold nanoparticle with detection antibody / detection antigen; performing ELISA detection to form the compound of capture antibody / capture antigen, to-be-detected antigen / to-be-detected antibody, detection antibody / detection antigen and gold nanoparticle; performing silver platinum dyeing and wrapping a silver shell layer and a platinum shell layer on the surface of the gold nanoparticle, thereby acquiring the nanometer enzyme Au@AgPt particle with peroxidase activity; utilizing the nanometer enzyme Au@AgPt particle to catalyze a peroxidase enzyme substrate to acquire the colored product so as to detect the quantity of the to-be-detected antigen / to-be-detected antibody. The method of forming enzyme after modifying is adopted, so that the method has the advantages of simplicity, quickness, convenience and high stability; the reduction of the catalytic activity of nanometer enzyme particle caused by nonspecific adsorption and modification can be effectively avoided; and therefore, a new platform is supplied for environment monitoring and disease diagnosis.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a nanozyme-based ELISA detection method, and more particularly relates to an ELISA detection method based on a nanozyme with peroxidase activity. Background technique [0002] Peroxidase is a protease that catalyzes the oxidation of electron donors in the presence of hydrogen peroxide. At present, peroxidase has been widely used in chemical sensing, environmental detection and disease diagnosis, for example, horseradish peroxidase (HRP)-labeled detection antibody can be used for ELISA detection. However, proteases have disadvantages such as easy inactivation, storage and preparation, which greatly limit the application range of peroxidases. [0003] Nanozymes are nanomaterials with catalytic ability similar to proteases. They have the advantages of simple synthesis, low cost and strong catalytic activity, which can well make up for the deficiency of pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58G01N33/543B01J31/06
CPCG01N33/543G01N33/581B01J23/52B01J31/06B01J35/23B01J35/50
Inventor 杨朝勇刘芳李久兴何梦逸朱志
Owner XIAMEN UNIV
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