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Homogeneous immunoassay based method for synchronous fluorescence detection of multiple disease markers

A synchronous fluorescence and marker technology, applied in the interdisciplinary field, can solve the problem of inability to realize the simultaneous detection of multiple components, and achieve the effect of sensitive and selective detection, good selectivity and strong versatility

Active Publication Date: 2013-03-13
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In recent years, fluorescence analysis has been widely used in virus detection due to its high sensitivity, rapidity and simplicity. However, traditional fluorescence analysis can only detect a single analyte and cannot simultaneously detect multiple components; Simultaneous detection of multiple components, the applicant carried out research on the simultaneous fluorescence scanning method, and established a simple, rapid, and simultaneous fluorescent detection method for homogeneous immunoassays of multiple antigens or viruses

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  • Homogeneous immunoassay based method for synchronous fluorescence detection of multiple disease markers
  • Homogeneous immunoassay based method for synchronous fluorescence detection of multiple disease markers
  • Homogeneous immunoassay based method for synchronous fluorescence detection of multiple disease markers

Examples

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Embodiment 1

[0029] A method for detecting multiple disease markers based on homogeneous immunosynchronous fluorescence, the steps of which are as follows:

[0030] (1) The monoclonal antibodies of influenza viruses H1N1, H5N1 and H9N2 to be tested were coupled with three succinimide ester-modified fluorescent dyes Alexa Fluor with different emission wavelengths, separated and purified, dissolved in pure water, and prepared as The concentrations of the three conjugates were all 1.0×10 -4 mol / L solution A is a mixed solution of three conjugates;

[0031] (2) Dissolve graphene oxide in pure water to make 1.0×10 -5 mol / L solution B;

[0032] (3) Mix 6 μL of solution A with a certain amount of solution B, and then dilute to 600 μL with pure water to obtain solution C. The dosage ratio of solution A and solution B is based on the molar ratio of the total amount of the three conjugates to graphene oxide 1 :10 meter;

[0033] (4) Take 500-1000 μL of solution C in a micro-volume fluorescence c...

Embodiment 2

[0040] A method for detecting multiple disease markers based on homogeneous immunosynchronous fluorescence, the steps of which are as follows:

[0041](1) The liver cancer markers AFP, CEA and CA125 monoclonal antibodies were coupled with three succinimide ester-modified fluorescent dyes Cy3 with different emission wavelengths, separated and purified, and dissolved in 15mM phosphate buffer solution of pH7.4 , the concentration of the three conjugates was prepared to be 1.0×10 -8 mol / L solution A is a mixed solution of three conjugates;

[0042] (2) Dissolve the mixture of graphene oxide and graphene in pure water to prepare a total concentration of 1.0×10 -6 mol / L solution B;

[0043] (3) Mix 6 μL of solution A with a certain amount of solution B, and then dilute to 600 μL with 15 mM pH 7.4 phosphate buffer solution to obtain solution C. The dosage ratio of solution A and solution B is based on the total amount of the three conjugates and The molar ratio of graphene oxide a...

Embodiment 3

[0049] A method for detecting multiple disease markers based on homogeneous immunosynchronous fluorescence, the steps of which are as follows:

[0050] (1) The monoclonal antibodies of anthrax virus, AIDS virus and hemorrhagic fever virus were respectively coupled with three succinimide ester-modified fluorescent dyes Cy5 with different emission wavelengths, separated and purified, dissolved in pure water, and prepared into three The concentration of each conjugate was 1.0×10 -5 mol / L solution A is a mixed solution of three conjugates;

[0051] (2) Dissolve graphene in pure water to make 1.0×10 -6 mol / L solution B;

[0052] (3) Mix 6 μL of solution A with a certain amount of solution B, and then dilute to 600 μL with 15 mM pH 7.4 phosphate buffer solution to obtain solution C. The dosage ratio of solution A and solution B is based on the total amount of the three conjugates and The graphene molar ratio is 1:20;

[0053] (4) Take 500-1000 μL of solution C in a micro-volume ...

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Abstract

The invention relates to interdisciplinary fields of biology, medicine, material, chemistry and virology, and discloses a homogeneous immunoassay based method for synchronous fluorescence detection of multiple disease markers. The method comprises the specific steps of: first, covalently coupling different fluorescent dyes with antibodies of different disease markers, and dissolving in water or other solvents to obtain a reaction solution A; second, mixing the solution A with a graphene oxide solution to obtain a reaction solution B; third, mixing the reaction solution A, the reaction solution B and water or other solvents to obtain a solution C; and fourth, adding an antigen solution into the mixed solution C, transferring the mixed solution to a fluorescence cuvette, measuring synchronous fluorescence intensity, and conducting quantitative analysis and multi-index analysis and diagnosis. The method has advantages of simpleness, rapidness, high sensitivity, high specificity and low cost, and can realize high-throughput detection, multiple index analysis diagnosis and remote diagnosis.

Description

technical field [0001] The invention relates to the interdisciplinary fields of biology, medicine, materials, chemistry, virology and other disciplines, and more specifically relates to a method for detecting multiple disease markers based on fluorescent dyes and graphene oxide homogeneous immuno-synchronous fluorescence. Background technique [0002] Protein arrays are usually used to detect multiple disease markers or viruses. Although this method has good selectivity, it has many steps, is time-consuming, laborious, and costly, and it cannot detect proteins or viruses of different properties at the same time. Using a homogeneous immune response for virus detection and disease diagnosis has the advantages of rapidity, sensitivity, and specificity, and can simultaneously detect multiple proteins or viruses with different properties. Symptoms of many diseases caused by viral infections are very similar, and it is impossible to determine which viral infection is the cause of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N21/64
Inventor 何治柯陈璐向东山
Owner WUHAN UNIV
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