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Fluorescence immunoassay method based on carbon quantum dots

A technology of carbon quantum dots and fluorescence, applied in the field of fluorescence immunoassay based on carbon quantum dots, can solve the problems of undeveloped application potential and few carbon dots

Inactive Publication Date: 2019-05-21
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the application of carbon dots in immunoassays is less, and its application potential in immunoassays remains to be developed

Method used

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  • Fluorescence immunoassay method based on carbon quantum dots
  • Fluorescence immunoassay method based on carbon quantum dots
  • Fluorescence immunoassay method based on carbon quantum dots

Examples

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Embodiment 1

[0072] The synthesis of embodiment 1 fluorescent carbon quantum dots

[0073] 5 g of citric acid and 7 g of thiourea were added to 15 mL of water. Stir continuously to dissolve the compound, place the mixed solution in a microwave reactor, and heat it at 800W for 7 minutes until the solution changes from colorless to dark brown solid, indicating that the reactant is carbonized and carbon quantum dots are formed. After cooling to room temperature, 25 mL of water was added to dissolve the product. The prepared carbon quantum dots were filtered through a 0.22 μm membrane to remove large particles, and dialyzed in ultrapure water for at least 24 h using a dialysis membrane (3500 Da) to remove unreacted small molecules. The prepared carbon quantum dot solution was separated and purified by Sephadex G25 column chromatography, and the collected carbon quantum dot solution with a maximum excitation wavelength of 370 nm and a maximum emission wavelength of 520 nm was named CDs-1.

[...

Embodiment 2

[0076] Embodiment 2 Amantadine fluorescent immunoassay detection

[0077] 1) Dilute the amantadine artificial antigen with coating solution to a concentration of 3.3×10 –5 mg / mL, add 100 μL / well into a 96-well ELISA plate, incubate at 4°C for 12 hours, then wash the plate with washing solution;

[0078] 2) Add blocking solution at 150 μL / well for blocking, incubate at 37°C for 1 hour, and wash the plate with washing solution;

[0079] 3) Add specific anti-amantadine antibody (concentration: 2.5×10 –5 mg / mL) and 50 μL / well of the sample solution to be tested, incubated at 37°C for 0.5 h, the antigen-antibody specific binding reaction occurs, and then the plate is washed with washing solution;

[0080] 4) Add horseradish peroxidase or alkaline phosphatase-labeled secondary antibody (concentration: 0.1 mg / mL) at 100 μL / well, incubate at 37 °C for 0.5 h to form enzyme-labeled immune complexes, and wash the plate with washing solution;

[0081] 5) Add horseradish peroxidase substr...

Embodiment 3

[0086] Embodiment 3 Aflatoxin B1 fluorescence immunoassay detection

[0087] 1) Dilute the artificial antigen of aflatoxin B1 with the coating solution to a concentration of 2.3×10 –5 mg / mL, add 100 μL / well into a 96-well ELISA plate, incubate at 4°C for 12 hours, then wash the plate with washing solution;

[0088] 2) Add blocking solution at 150 μL / well for blocking, incubate at 37°C for 1 hour, and wash the plate with washing solution;

[0089] 3) Add specific anti-aflatoxin B1 antibody (concentration: 2.1×10 –5 mg / mL) and 50 μL / well of the sample solution to be tested, incubated at 37°C for 0.5 h, the antigen-antibody specific binding reaction occurs, and then the plate is washed with washing solution;

[0090] 4) Add horseradish peroxidase or alkaline phosphatase-labeled secondary antibody (concentration: 0.1 mg / mL) at 100 μL / well, incubate at 37 °C for 0.5 h to form enzyme-labeled immune complexes, and wash the plate with washing solution;

[0091] 5) Add horseradish p...

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Abstract

The invention provides a fluorescence immunoassay method based on carbon quantum dots. The carbon quantum dot is prepared by using citric acid as a carbon source and thiourea or urea as a nitrogen source. The carbon quantum dot is applied to an enzyme-linked immunoassay method. A novel high-sensitivity fluorescence immunoassay method based on carbon quantum dots is established. An efficient fluorescence internal filtration effect is generated between carbon quantum dots and an enzyme-catalyzed chromogenic substrate, and an absorbance signal formed by reaction of horse radish peroxidase / alkaline phosphatase catalytic substrate in enzyme-linked immunoassay can be successfully and effectively converted into a fluorescence signal. The method has the advantages of simplicity in operation, low cost, high sensitivity, good stability and the like, and can be used for quantitative detection of the micromolecular hapten. In addition, the method has good expansibility and is expected to be expanded to other target detection systems.

Description

technical field [0001] The invention relates to the technical field of antigen detection, in particular to a fluorescent immunoassay method based on carbon quantum dots. Background technique [0002] Immunoassay is a kind of rapid analysis technology based on specific recognition and binding between antigen and antibody. Immunoassay can be applied to the determination of macromolecular compounds (such as proteins, bacteria, etc.) and small molecular compounds (such as pesticides, veterinary drugs, mycotoxins, etc.). Due to the advantages of high specificity, fast detection speed, low cost and suitable for on-site screening of a large number of samples, immunoassay has been widely used in analytical fields such as food safety detection and environmental monitoring. [0003] Enzyme-linked immunoassay method is a commonly used immunoassay method. It has the advantages of low instrument dependence, simple operation, low cost, strong specificity, high sensitivity, large-scale de...

Claims

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Application Information

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IPC IPC(8): G01N33/533G01N33/535G01N33/537
Inventor 沈建忠王战辉温凯江海洋董保磊段长飞于雪芝史为民
Owner CHINA AGRI UNIV
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