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Production method for enzyme functionalized nano immunity marker and use thereof

An immunolabeling and functionalization technology, which is applied in the direction of chemiluminescence/bioluminescence, analysis by causing chemical reactions of materials, and electrochemical variables of materials, etc., can solve the problems of amplification, environmental pollution, influence of experimental steps, etc., and improve sensitivity , enhanced chemiluminescent performance, improved sensitivity and reproducibility

Inactive Publication Date: 2013-05-29
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A variety of biological and nanotechnology have been used for signal amplification of biomolecular detection, such as polymerase chain reaction (PCR), enzyme catalysis, signal amplification of metal nanoparticles and quantum nanoparticles, etc. These methods require special instruments or techniques To assist in the detection, the amplification of the enzyme is also easily affected by environmental pollution and cumbersome experimental steps

Method used

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  • Production method for enzyme functionalized nano immunity marker and use thereof
  • Production method for enzyme functionalized nano immunity marker and use thereof
  • Production method for enzyme functionalized nano immunity marker and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Preparation of enzyme-functionalized nano-immunolabels

[0023] Weigh 0.2 g of silica nanospheres with a particle size of 100±2 nm, add it into 5 mL of 5% GPMS toluene solution, and stir and react at room temperature for 24 h. After the reaction, the silica microspheres were centrifuged (rotating at 12000r / min), and the surface-adsorbed GPMS was cleaned with toluene and absolute ethanol respectively, and then heated to 100°C and dried under the protection of nitrogen to obtain surface-modified Epoxy-functional silica microspheres.

[0024] Take 15 mg of epoxy-functionalized silica microspheres, add them to a mixed solution of 450 μL 1 mg / mL HRP and 50 μL 12.7 μg / mL anti-AFP antibody, stir and react at 4 °C for 24 h, centrifuge and use phosphoric acid containing Wash with salt buffer solution three times to remove the adsorbed HRP and anti-AFP antibodies, and then treat the modified silica microspheres with 500 μL of 1% BSA for 3 hours to seal the residual active epoxy ...

Embodiment 2

[0026] Sandwich immunization process

[0027] Soak the clean gold pieces in 100 μL of 10 mM 1:1 MUA / MU ethanol solution for 15 hours, rinse with distilled water, activate in a mixed solution of 10 mM EDC and 10 mM NHS for 30 minutes, and then immerse in anti -incubating in the AFP primary antibody solution for 1 hour to obtain an anti-AFP primary antibody-modified gold electrode.

[0028] The above-mentioned anti-AFP primary antibody modified gold electrode was soaked in solutions containing different concentrations of AFP antigen and incubated for 30 minutes to capture the AFP antigen by immune reaction, and then place the gold sheet on 15 mg / mL enzyme functionalized nano-immunolabel ( Prepared in Example 1) and incubated in the suspension for 30 minutes, using the immunoreaction between the anti-AFP secondary antibody modified on the surface of the marker and the AFP antigen captured on the surface of the gold electrode, the enzyme functionalized nano-immunomarker was captured...

Embodiment 3

[0030] immunoassay

[0031] (1) Electrochemical detection method: the gold electrode modified by the sandwich immune process obtained in the above-mentioned embodiment 2 is used as the working electrode, the platinum wire is used as the auxiliary electrode, and the saturated calomel electrode is used as the reference electrode. h 2 o 2 , pH 7.0 phosphate solution, at 50mV s -1 The scanning speed is cyclic scanning, because the modified HRP on the electrode catalyzes the H 2 o 2 The reduction reaction, and the reduction current response increases with the increase of the amount of HRP on the electrode, and the amount of HRP on the electrode is directly related to the concentration of AFP antigen in the solution, so the change of the amount of HRP on the detection electrode can reflect the solution Antigen concentration is medium, and because the present invention uses silica pellets as the carrier of the enzyme, the amount of enzyme loaded in a single immune process is grea...

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Abstract

The invention relates to a preparation technology for an enzyme functionalized nanometer immune label. The surface of a silicon dioxide nanometer microballoon is epoxidized by utilizing the silanization reaction of the hydroxide radical on the surface of the silicon dioxide nanometer microballoon and GPMS, and then a silicon dioxide microballoon immune label which is modified together by horse radish peroxidase and an anti-AFP second antibody is obtained by utilizing the reaction of an epoxy group and active amino-groups in the horse radish peroxidase and anti-AFP molecules. The surface of the microballoon is rich of enzymes so that detection signals in a single immune process are enlarged, thereby the sensitivity of the detection of low-concentration biomolecules is improved. Meanwhile, the grain diameter and the surface height of the used silicon dioxide microballoon are both uniform so that the enzymes loaded on a single silicon dioxide microballoon are almost same as the biomolecules in amount, so that the repeatability for detection is greatly improved.

Description

1. Technical field [0001] The present invention relates to a preparation technology of enzyme-functionalized nano-immunomarkers, in particular to a preparation method of silica microsphere immunomarkers co-modified by horseradish peroxidase (HRP) and a second antibody (Ab2) , and an electrochemical method for low-concentration biomolecular detection using the enzyme-functionalized nano-immunolabel. 2. Background technology [0002] Existing technology: With the development of biotechnology and the advancement of bioinformatics, the current methods of early clinical diagnosis, curative effect observation and prognosis monitoring are becoming more and more perfect, and many molecular markers with specific targets have been discovered. However, in the early stages of disease development, the expression levels of these tumor-associated antigens or tumor markers are very low, or some other specific cytokines are produced, but due to the limitations of detection methods, sensitivi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/535G01N27/26G01N21/76
Inventor 刘松琴吴亚锋陈成良
Owner SOUTHEAST UNIV
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