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A method for detecting hydrogen peroxide and peroxidase based on modified gold nanoparticles

A technology of peroxidase and hydrogen peroxide, which is applied in the field of food safety detection technology, can solve the problems such as the unspecified detection range and sensitivity of hydrogen peroxide, and achieve high-sensitivity detection, convenient quality control, and mature modification technology Effect

Active Publication Date: 2019-10-29
SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sensitivity of this method is extremely high. However, its reaction is based on the catalysis of contact enzymes, which is different from the reaction catalyzed by the marker enzyme horseradish peroxidase, which is commonly used in immunoassays. The application of this scheme requires the modification of the marker enzymes, which is not suitable for the existing immunoassays. Reagent kits require major reagent replacement
In addition, there are also reports (Hui L., WeipingQ.et.al.Analytical Chemistry.2009, 80(21), 8916-8922) directly using tetrachloroalloy acid ions to oxidize hydrogen peroxide, making it adsorbed on silica microspheres The gold nanoparticles on the surface grow, and the surface plasmon resonance changes, and the degree of change is related to the concentration of hydrogen peroxide. However, the paper does not give the detection range and sensitivity of direct detection of hydrogen peroxide.
The hydrogen peroxide detection scheme based on modified gold nanoparticles has the advantages of good repeatability, convenient quality control, and high sensitivity, and has not yet been reported in the literature

Method used

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  • A method for detecting hydrogen peroxide and peroxidase based on modified gold nanoparticles

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Experimental program
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Effect test

Embodiment 1

[0022] 1) Preparation of p-mercaptophenol modified nano-gold sol:

[0023] Nano-gold was prepared by citric acid reduction method, vitamin C reduction method, sodium borohydride reduction method and white phosphorus reduction method, and the concentration of the prepared nano-gold solution was measured by spectrophotometry.

[0024] Add 100mL of 10nM nano-gold solution with a particle size of 13nm to 1mL of 20μM p-mercaptophenol solution. After mixing evenly, wrap it in tin foil and shake slowly for 2 hours to obtain p-mercaptophenol-modified nano-gold sol, and observe it with a transmission electron microscope. The mercaptophenol-modified nano-gold sol, and the mercapto-phenol-modified nano-gold sol were observed under a transmission electron microscope. It can be seen that there is no aggregation after the nano-gold modification, and it can be used as a detection object.

[0025] 2) Response of mercaptophenol-modified nano-gold sol to hydrogen peroxide:

[0026] Add 5 μL of...

Embodiment 2

[0028] 1) The preparation of p-mercaptophenol modified nano-gold sol is the same as in Example 1;

[0029] 2) establish the standard curve of hydrogen peroxide:

[0030] a. Add ten-fold gradient diluted hydrogen peroxide solution to the 7 microwell plates of the microplate reader, and then add 5 μL 3 μM peroxidase solution, 5 μL volume percent concentration 0.3% H 2 o 2 solution and 200 μL p-mercaptophenol-modified nano-gold sol, as 7 experimental groups, in another microwell plate, add 40 μL water, 5 μL 3 μM peroxidase solution, 200 μL p-mercaptophenol-modified nano-gold sol, as a negative control group , reacted for 10 minutes, and added sodium chloride solution to a final concentration of 30 mM. Detecting the optical densities at wavelengths of 520nm and 720nm of the p-mercaptophenol-modified nano-gold sol containing hydrogen peroxide of known concentration in the experimental group;

[0031] b. Detect the detection results of hydrogen peroxide solutions with different k...

Embodiment 3

[0035] 1) Preparation of p-mercaptoaniline modified nano-gold sol:

[0036] Nano-gold was prepared by citric acid reduction method, vitamin C reduction method, sodium borohydride reduction method and white phosphorus reduction method, and the concentration of the prepared nano-gold solution was measured by spectrophotometry.

[0037] Add 100mL of 10nM nano-gold solution with a particle size of 13nm to 1mL of 80μM p-mercaptoaniline solution, mix evenly, wrap it in tin foil and shake slowly for 2 hours to obtain p-mercaptoaniline-modified nano-gold sol;

[0038] 2) Response of mercaptoaniline-modified nano-gold sol to hydrogen peroxide:

[0039] Add 5 μL of 0.3% HO to 5 μL of 3 μM peroxidase solution 2 o 2 solution, 200 μL p-mercaptophenol modified nano-gold sol, reacted for 10 minutes, and added sodium chloride solution to a final concentration of 30 mM. As a control sample, 200 μL of p-mercaptoaniline-modified nano-gold sol was added to 5 μL of 3 μM peroxidase solution, rea...

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Abstract

The invention provides a modified nanogold-based hydrogen peroxide and peroxidase detection method. The method comprises the following steps: 1, modifying: adding a p-mercaptophenol or p-aminothiophenol solution into a nanogold solution to modify the nanogold, mixing uniformly, coating with a tin foil, and placing in a dark place to obtain modified nanogold particle sol; 2, establishing a standard curve; 3, detecting a to-be-detected sample: taking an actual hydrogen peroxide sample or a peroxidase-containing sample as a detected object and the modified nanogold particle sol as a detecting object, detecting the optical density of the modified nanogold particle sol containing the actual hydrogen peroxide sample or containing the actual peroxidase sample, and comparing the ratio of the optical density with the standard curve to obtain the concentration or the enzyme activity concentration of the actual hydrogen peroxide sample or the actual peroxidase-containing sample. The method is capable of performing supersensitive detection on the hydrogen peroxide and peroxidase, is low in cost and has the advantage of high detection flux.

Description

technical field [0001] The invention relates to a method for supersensitive detection of hydrogen peroxide and peroxidase by modifying nano-gold particles, and then detects enzymes and substrates produced by hydrogen peroxide in various reaction processes, and can be used for peroxidase The immunological detection of the marker belongs to the fields of food safety detection technology, biochemical and immunodiagnosis. Background technique [0002] Nano-gold particles, also known as nano-gold sol or colloidal gold, refer to gold nano-spherical structures below 200 nanometers. It has the characteristics of high electron density, localized surface plasmon resonance (Localized Surface Plasmon Resonance) and strong absorption of visible light with a wavelength of about 520 nanometers. When its aggregation or shape changes, its surface plasmon absorption peak can change significantly. Using this feature, small molecule interactions can be designed to cause changes in the aggregat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/59G01N33/535G01N33/543
CPCG01N21/5907G01N33/535G01N33/54346
Inventor 梁晓声
Owner SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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