Modified nanogold-based hydrogen peroxide and peroxidase detection method

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

Active Publication Date: 2017-11-24
SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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  • Abstract
  • Description
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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 aci

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  • Modified nanogold-based hydrogen peroxide and peroxidase detection method

Examples

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Comparison scheme
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Example Embodiment

[0021] Example 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 10nM gold nano-gold solution with a particle size of 13nm to 1mL 20μM p-mercaptophenol solution. After mixing, the tin foil is coated and shaken slowly for 2 hours to obtain p-mercaptophenol-modified gold nano sol, which is observed by transmission electron microscope. The p-mercaptophenol-modified nano-gold sol and the transmission electron microscopy observation of the mercaptophenol-modified nano-gold sol show that there is no aggregation after the modification of the nano-gold, and it can be used as a detection substance.

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

[0026] In 5...

Example Embodiment

[0027] Example 2

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

[0029] 2) Establish a standard curve of hydrogen peroxide:

[0030] a. Add ten-fold dilution of hydrogen peroxide solution to the 7 microplates of the microplate reader, and then add 5μL of 3μM peroxidase solution, 5μL volume percentage concentration of 0.3% H 2 O 2 Solution and 200μL p-mercaptophenol modified gold nano sol, as 7 experimental groups, in another microplate, add 40μL water, 5μL 3μM peroxidase solution, 200μL p-mercaptophenol modified gold nano sol, as a negative control group , React for 10 minutes, add sodium chloride solution to the final concentration of 30mM. Detect the optical density at wavelengths of 520nm and 720nm of p-mercaptophenol modified nano-gold sol containing known concentration of hydrogen peroxide in the experimental group;

[0031] b. The detection results of different known concentrations of hydrogen peroxide solution are obtained....

Example Embodiment

[0034] Example 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 100 mL of 10nM gold nano-gold solution with a particle size of 13nm to 1 mL of 80μM p-mercaptoaniline solution, mix well, and coat with tin foil and shake slowly for 2 hours to obtain p-mercaptoaniline modified gold nano sol;

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

[0039] In 5μL of 3μM peroxidase solution, add 5μL of 0.3% H 2 O 2 Solution, 200 μL p-mercaptophenol modified gold nano sol, react for 10 minutes, add sodium chloride solution to a final concentration of 30 mM. The control sample was 5 μL of 3 μM peroxidase solution and 200 μL of p-mercaptoaniline modified nano-gold sol,...

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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 present invention relates to a method for supersensitive detection of hydrogen peroxide and peroxidase by modified gold nanoparticles, and then detects the enzymes and substrates produced by hydrogen peroxide during various reactions, and can be used for peroxidase Labeled immunoassays belong to the fields of food safety testing technology, biochemistry and immunodiagnosis. Background technique [0002] Nano gold particles, also called nano gold sol or colloidal gold, refer to gold nano-spherical structures below 200 nanometers. It has high electron density, localized surface plasmon resonance (Localized Surface Plasmon Resonance) has strong absorption of visible light with a wavelength of about 520 nm. When its aggregation or morphology changes, its surface plasmon absorption peak can be significantly changed. Using this feature, the interaction of small molecules can be designed to change the aggregation state of nano-gold, and then the corresponding...

Claims

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Application Information

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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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