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A method for detecting alpha-fetoprotein based on luminol functionalized magnetic immunoprobe

A functionalization technology of alpha-fetoprotein, applied in the direction of measuring devices, biological testing, material inspection products, etc., can solve the problems of difficult control of coating solid phase conditions, high cost, analysis errors, etc.

Inactive Publication Date: 2017-05-17
LINYI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Isolation of antibody-antigen immune complexes is often tedious and time-consuming when using these solid supports and is often a major source of analytical error
Due to the uneven immobilization of antibodies or antigens on the solid phase and the high cost, 10% of most antibodies or antigens can be adsorbed on the solid phase for the formation of antibody-antigen immune complexes, making it difficult to control the conditions of coating the solid phase

Method used

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  • A method for detecting alpha-fetoprotein based on luminol functionalized magnetic immunoprobe
  • A method for detecting alpha-fetoprotein based on luminol functionalized magnetic immunoprobe
  • A method for detecting alpha-fetoprotein based on luminol functionalized magnetic immunoprobe

Examples

Experimental program
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Effect test

Embodiment 1

[0015] Preparation of luminol functionalized magnetic immunoprobes (see figure 1 )

[0016] (1) Using carboxyl-modified magnetic microspheres to prepare luminol-functionalized magnetic immunoprobes.

[0017] (a) Wash 100 μL of 10 mg / mL carboxy-modified magnetic microspheres with 0.1 M imidazole-hydrochloric acid buffer solution at pH 7.0, add 0.1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiene amine and 0.1 M N-hydroxysuccinimide, incubated at 37°C for 5 hours, washed with 0.01M pH=7.4 phosphate buffer solution, and magnetically separated to obtain activated magnetic microspheres.

[0018] (b) 200 μL 1.0 × 10 -4 M luminol and 200 μL 0.02 mg / mL secondary antibody to alpha-fetoprotein were added to the activated magnetic microspheres, incubated in an incubator at 37 °C for 12 hours, and washed with 0.01M pH=7.4 phosphate buffer solution , adding 200 μL of 0.1% bovine serum albumin solution and incubating at 37 °C for 2 hours, washing with 0.01M pH=7.4 phosphate buffer solution...

Embodiment 2

[0026]Electrochemiluminescent detection of alpha-fetoprotein (see figure 2 )

[0027] (1) According to Example 1, a luminol-functionalized magnetic immunoprobe was prepared.

[0028] (2) According to Example 1, a gold electrode modified with an alpha-fetoprotein primary antibody was prepared.

[0029] (3) Drop 8 μL of the sample or standard to be tested on the surface of the gold electrode modified with primary antibody to alpha-fetoprotein, incubate at 37 °C for 2 hours, wash with 0.01 M pH=7.4 phosphate buffer solution, add luminol function dropwise The magnetic immunoprobe solution was incubated at 37 °C for 2 hours to form a primary antibody / antigen / secondary antibody / luminol sandwich structure on the surface of the gold electrode.

[0030] (4) Electrochemiluminescence detection. The prepared gold electrode was immersed in 2.0 mL electrochemiluminescence reaction reagent, the platinum wire electrode was used as the counter electrode, and the Ag / AgCl electrode was used ...

Embodiment 3

[0032] According to Example 1 and Examples, the luminol functionalized magnetic immunoprobe prepared by this method was used to detect alpha-fetoprotein in 5 human serum samples, and the results were compared with the traditional enzyme-linked immunoassay, and the correlation was drawn. curve. The results show a good correlation between the two (see Figure 4 ).

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Abstract

The invention provides a method for detecting alpha fetoprotein by using a magnetic immune probe based on luminol functionalization, and belongs to the technical fields of nano functional materials, bio-sensing techniques and electrochemistry. A magnetic microsphere is large in specific surface area, strong in electric conducting capacity and convenient to separate. A luminol functionalized magnetic immune probe is produced by preparing a luminol functionalized magnetic microsphere and labelling an alpha fetoprotein second antibody by using the luminol functionalized magnetic microsphere. An 'alpha fetoprotein first antibody / alpha fetoprotein antigen / luminol functionalized magnetic immune probe' type composite structure is constructed on the surface of a gold electrode, and the sensitive and specific detection on the alpha fetoprotein antigen can be achieved by detecting an electrochemiluminescence signal. An enzyme labelled antibody method which is commonly used at present is complex in probe preparation and operation and is easy to reduce the enzyme activity; and compared with the enzyme labelled antibody method, a luminol functionalized magnetic immune probe labelled antibody is simple and convenient to prepare and easy to store, and can be used for detecting the alpha fetoprotein antigen.

Description

technical field [0001] The present invention relates to the preparation of luminol-bonded magnetic immunoprobes. The traditional enzyme labeling method to prepare probes requires complicated separation steps and easy to reduce enzyme activity. The method is convenient for separation, easy to operate and easy to store, and realizes the detection of alpha-fetoprotein by electrochemiluminescence. Background technique [0002] Biofunctional substances with molecular recognition capabilities, such as enzymes, antigens, antibodies, cells, etc., are wrapped or adsorbed on certain polymer materials, biopolymers or inorganic materials to prepare sensors, which are called immobilization of biofunctional substances. change. The immobilization method of biomolecules is the key to the development of immunosensing technology, and it has become one of the most effective ways to improve the stability, sensitivity and selectivity of immunoassay methods, and represents the current developme...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/76G01N33/68G01N33/553
Inventor 李雪梅郭英姝张书圣
Owner LINYI UNIVERSITY