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A kind of ELISA detection method based on nanozyme with catalase activity

A technology of catalase and detection method, which is applied in the field of ELISA detection of nanozyme, can solve the problems of reduced catalytic activity and unstable catalytic activity of nanozyme, achieve strong catalytic activity, avoid the decline of enzyme catalytic activity, and have good stability Effect

Active Publication Date: 2017-11-17
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • A kind of ELISA detection method based on nanozyme with catalase activity
  • A kind of ELISA detection method based on nanozyme with catalase activity
  • A kind of ELISA detection method based on nanozyme with catalase activity

Examples

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Effect test

Embodiment 1

[0040] Example 1: Preparation of gold nanoparticles

[0041] 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

[0042] Embodiment 2: the comparative experiment of silver platinum dyeing condition

[0043] 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.

[0044] 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 .

[0045] Subseq...

Embodiment 3

[0048] Example 3: Characterization of nanozyme Au@AgPt particles with catalase activity obtained after silver-platinum staining

[0049] Add AgNO to the gold nanoparticles AuNPs obtained in 1mL 2.5nM Example 1 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. Washed three times with 300 μL water to obtain Au@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 0.5 mM and 10 mM, respectively, reacted at 37 °C for 20 min, and washed three times with 300 μL water to obtain nanozyme Au@AgPt particles with catalase 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.

[0050] The result is as image 3 As shown...

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Abstract

The invention discloses an ELISA detection method based on nano-enzyme with catalase activity. The ELISA detection method is applicable to a double antibody sandwich method or a dual antigen sandwich method. The method comprises the following steps: firstly modifying detection antibody / detection antigen on a gold nanoparticle; performing ELISA detection and forming a compound of capturing antibody / capturing antigen, to-be-detected antibody / to-be-detected antigen, detection antibody / detection antigen and gold nanoparticle; performing silver platinum dyeing and wrapping the gold nanoparticle surface with silver shell layer and platinum shell layer, thereby acquiring the nano-enzyme Au@AgPt grain with catalase activity; and utilizing Au@AgPt grain to catalyze hydrogen peroxide decomposition for generating hydrogen in an airtight system, increasing the air pressure in the airtight system, detecting the air pressure change, namely, detecting and acquiring the volume of the to-be-detected antibody / to-be-detected antigen. A method of forming enzyme after modifying is applied to the ELISA detection method, so that the ELISA detection method has the advantages of simplicity and quickness, high stability, capability of effectively preventing the nano-enzyme particle catalytic activity caused by nonspecific adsorption and modification from decreasing and capability of supplying new platform to the 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 catalase activity. Background technique [0002] Catalase is a protease that catalyzes the decomposition of hydrogen peroxide to produce oxygen. At present, catalase has been widely used in chemical sensing, environmental detection and disease diagnosis. However, protease has disadvantages such as easy inactivation, difficult storage and difficult preparation, which greatly limits the application range of catalase. [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 proteases. Among various nanozymes, platinum nanoparticles are widely used in environmental monitoring and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/573G01N33/532
CPCG01N33/532G01N33/573G01N2333/908
Inventor 杨朝勇李久兴刘芳何梦逸朱志
Owner XIAMEN UNIV
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