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Glycoprotein dynamic light scattering immunization method based on phenylboronic acid cross-linking agent

A dynamic light scattering and glycoprotein technology, applied in the field of immunoassay, can solve the problems of false positive signal, low sensitivity, unstable signal, etc., and achieve the effect of simple operation steps, high sensitivity detection, and reduced demand.

Active Publication Date: 2021-11-23
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the deficiencies in the prior art, the present invention provides a glycoprotein dynamic light scattering immunization method based on a phenylboronic acid cross-linking agent, which saves an antibody solution The problem of difficult preparation of paired antibodies has realized the advantages of high sensitivity and timely detection of glycoproteins, and solved the traditional enzyme-linked immunosorbent assay, which is the main detection method of existing glycoproteins, with complicated detection steps, low antigen-antibody utilization and low reaction efficiency , low sensitivity, signal instability and false positive signals, etc.

Method used

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  • Glycoprotein dynamic light scattering immunization method based on phenylboronic acid cross-linking agent
  • Glycoprotein dynamic light scattering immunization method based on phenylboronic acid cross-linking agent
  • Glycoprotein dynamic light scattering immunization method based on phenylboronic acid cross-linking agent

Examples

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Effect test

Embodiment 1

[0040] Example 1 Application of using magnetic nano-microspheres to detect the content of alpha-fetoprotein

[0041] 1 Preparation of carboxylated Fe3O4 magnetic microspheres

[0042] 1) Preparation of oleated Fe3O4 magnetic beads

[0043] a. Synthesis of Fe304 magnetic beads, add 300mL ultrapure water into a 500mL three-necked flask, pass through N2 to remove oxygen in the water, and preheat at 50°C for 20min, add 3.2gFeCl2·H20, 5.2gFeCl3 Magnetic stirring and mixing, then add 25mL Ammonia water, the yellow solution quickly turns black, react at a constant temperature of 50°C for 30 minutes, separate the synthesized magnetic beads by magnetic suction, wash 3-5 times with ultrapure water until the pH of the solution reaches neutral after magnetic absorption, and then redissolve in 300mL ultrapure water middle.

[0044] b. Oil acidification modification of Fe304 magnetic beads, add the magnetic beads in the previous step into a three-necked flask, pass through N2, add 2.4mL o...

Embodiment 2

[0059] Example 2 Glycoprotein-alpha-fetoprotein as the object to be detected

[0060] 6 μL of alpha-fetoprotein detection antibody-labeled Fe304 microsphere probe was reacted with 200 μL of different concentrations of alpha-fetoprotein samples for 5 minutes, magnetically sucked for 5 minutes, the supernatant was discarded and ultrasonically washed 2-3 times with PB7.5 (0.01mol / L), and then added 800μL of 0.2mg / mL 8arm PEG@3-APBA was reconstituted and sonicated, incubated at 25°C for 5min, taken out at 25°C and measured with a Malvern Nanoparticle Size Analyzer to measure the average hydration kinetic diameter of the solution, and calculated the average offspring Enter the standard curve to obtain the concentration of alpha-fetoprotein in the sample to be tested. The specific experimental results are as follows: the linear standard curve is y=19.68ln(x)+246, R=0.9875, see the attached figure 2 , the lowest detection limit of the method is defined as the average hydration parti...

Embodiment 3

[0061] Example 3 Application of gold magnetic microspheres to detect alpha-fetoprotein content

[0062] 1 Preparation of gold magnetic probe labeled with detection antibody

[0063] Take 10 μL of gold magnetic nanoparticles (10 mg / mL) and add them to 200 μL of pH8.0 PB, add 4 μg of the detection antibody corresponding to alpha-fetoprotein, stir and react at room temperature for 30 min; continue to stir the reaction, add 0.2 μg of 1-ethyl-(3-di After methylaminopropyl)-3-ethylcarbodiimide hydrochloride, stir at room temperature for 30 minutes, add twice, after the completion of the reaction, magnetic suction for 5 minutes, discard the supernatant, and then add acid with a mass volume fraction of 2% for hydrolysis casein, and added 0.2 μg 1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, stirred at room temperature for 1 h, and discarded the supernatant after magnetic absorption for 5 min, then added The 6-amino-3-pyridine phenylboronic acid with a mass volume ...

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Abstract

The invention relates to a glycoprotein dynamic light scattering immunization method based on a phenylboronic acid cross-linking agent. The phenylboronic acid cross-linking agent and the magnetic carrier marked by the immune recognition element are used as a dynamic light scattering signal enhancement probe to form a multilayer sandwich structure by taking target glycoprotein as a bridge, and the average hydration kinetic particle size change of the solution before and after the multilayer sandwich structure is used as a dynamic light scattering signal to be output; and the content of glycoprotein in the to-be-detected sample is determined by utilizing the diameter change of hydration kinetics. According to the glycoprotein dynamic light scattering immunization method based on the phenylboronic acid cross-linking agent, the operation steps are simple, high-sensitivity detection of glycoprotein in a complex sample can be achieved in a short time, the prepared immunomagnetic beads can separate and enrich target protein from a complex sample matrix, and interference of the sample matrix on follow-up detection is effectively eliminated. The problem that the paired antibody is difficult to prepare can be solved, the glycoprotein can be simply, conveniently, rapidly and timely detected, and the method is worthy of being further popularized and used.

Description

technical field [0001] The invention relates to the technical field of immune analysis, in particular to a glycoprotein dynamic light scattering immunological method based on a phenylboronic acid cross-linking agent. Background technique [0002] In recent years, boric acid as an important ligand has been widely used in the construction of functionalized materials, and has been widely used in sensors to detect biomolecules of cis-diols. Integrating important molecules containing cis-diol substances, the unique properties of boronic acid combined with cis-diol make the recognition molecule phenylboronic acid can be used as an antibody substitute for biomimetic immunological analysis, therefore, the recognition can be realized on the basis of saving an antibody Detection of cis-diol species (glycoproteins), thus greatly reducing the need for paired antibodies. [0003] Dynamic light scattering technology, also known as photon correlation spectroscopy or quasi-elastic light sc...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/68G01N21/47
CPCG01N33/54326G01N33/68G01N21/47
Inventor 黄小林熊勇华陈静胡佳琪朱康
Owner NANCHANG UNIV
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