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Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor

An immunosensor and probe technology, applied in the field of molecular recognition probes, can solve problems such as difficulty in overcoming the influence of non-specific adsorption, limited enrichment effect of secondary antibodies, and inability to update electrodes, thereby eliminating the need for centrifugation and washing processes. , not easy to inactivate, and the effect of low detection limit

Active Publication Date: 2011-11-23
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reported electrochemical amperometric sensors also have the following deficiencies in use: ① more "one-step method" is used, that is, the immune complex formed after the combination of antigen and antibody is used to cause the electrode surface current to drop, and the quantification is carried out according to the falling current, so that it is impossible It is difficult to avoid the influence of non-specific adsorption; ②When the antigen / antibody on the surface of the electrode is immunocombined, the product is difficult to remove, the electrode cannot be renewed, and the use is mostly disposable; ③Although the amperometric immunosensor has many advantages, it still faces Specific detection objects with very complex backgrounds (such as serum samples) often need to be tested after a certain sample purification process, which affects its ease of use
However, a single nanoparticle has limited enrichment effect on the secondary antibody

Method used

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  • Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor
  • Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor
  • Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1. Preparation and characterization of secondary antibody probes

[0030] (1) Preparation of magnetic nanoparticles

[0031] Preparation of magnetic nanoparticles: For specific preparation methods, please refer to the invention patent application with publication number CN 101302361A.

[0032] Characterization of Magnetic Nanoparticles: Fe by X-ray Fluorescence Spectroscopy (XRF) 3 o 4 / ZrO 2 To characterize, Zr-K appeared β (17.8keV), Zr-K α (15.8keV), Zr-L β (2.1ke), Zr-L α (2.0keV) peak and Fe-K β (7.1KeV), Fe-K α (6.4keV) peak, indicating that Zr and Fe elements exist in the magnetic particles.

[0033] (2) Preparation and characterization of antibody-loaded nanospheres

[0034] Preparation of antibody-loaded nanospheres: Disperse 10 mg of magnetic nanoparticles in 5 mL of pH 7.0 phosphate buffer, add 1 mg of horseradish peroxide-labeled alpha-fetoprotein secondary antibody (HRP-anti-AFP), and stir for 6 h , magnetically separated under magnetic condition...

Embodiment 2

[0058] Embodiment 2 (comparative experiment 1)

[0059] 1. Experimental materials

[0060] 1. Antigen and antibody: AFP antigen standard, alpha-fetoprotein monoclonal primary antibody (anti-AFP), horseradish peroxidase-labeled alpha-fetoprotein polyclonal antibody (HRP-anti-AFP) and AFP ELISA kit Both purchased Zhengzhou Bosai Biotechnology Co., Ltd.

[0061] 2. Amperometric immunosensor: prepared according to the method described in Example 1.

[0062] 3. Secondary antibody probe

[0063] 3.1. Sample: the secondary antibody probe prepared by the method described in Example 1.

[0064] 3.2. Reference substance

[0065] Control substance 1:

[0066] (1) Preparation of magnetic nanoparticles

[0067] Preparation of magnetic nanoparticles: For specific preparation methods, please refer to the invention patent application with publication number CN 101302361A.

[0068] (2) Preparation and characterization of antibody-loaded nanospheres

[0069] Preparation of antibody-load...

Embodiment 3

[0087] 1. Preparation and characterization of secondary antibody probes

[0088] (1) Preparation and characterization of magnetic nanoparticles

[0089] Shown in step 1 with embodiment 1.

[0090] (2) Preparation and characterization of antibody-loaded nanospheres

[0091] Preparation of antibody-loaded nanospheres: 10 mg of magnetic nanoparticles were dispersed in 5 mL of pH 7.0 phosphate buffer, and 1 mg of horseradish peroxidase-labeled HIV p24 antibody (HRP-anti-HIV p24) was added. After stirring for 6 hours, an external magnetic field was applied to separate the unbound antibody to obtain antibody-loaded nanospheres.

[0092] (3) Preparation of nanospheres

[0093] Shown in step 3 with embodiment 1.

[0094] (4) Preparation of secondary antibody probe

[0095] Shown in step 4 with embodiment 1.

[0096] 2. Use the secondary antibody probe to detect the antigen concentration

[0097] (1) Preparation of Amperometric Immunosensor

[0098] (a) Graphene / chitosan (GS / CS...

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Abstract

The invention relates to the field of biological monitoring, and specifically relates to a secondary antibody probe for a sandwich immune reaction by using an amperometric immunosensor, wherein, the secondary antibody probe is composed of calf thymus DNA molecular chains and nanospheres adhering to the calf thymus DNA molecular chains, and the surfaces of the nanospheres are covered by enzyme labeled antibody proteins while magnetic nanometer particles, which are formed by depositing a layer of amorphous zirconia on the surfaces of nanometer ferriferrous oxide particles, are filled in the nanospheres. The secondary antibody probe provided in the invention is suitable for measurring concentration of antigens by a sandwich immune reaction employing an amperometric immunosensor, and has the advantages of wide linear range of detection and low detection limits.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a molecular recognition probe, which is suitable for measuring the concentration of an antigen by using an ampere immunosensor for sandwich immunoreaction. Background technique [0002] In 1990, Henry et al. proposed the concept of immunosensors. Immunosensors use antigens and antibodies as molecular recognition elements, and convert the signals generated during the antigen-antibody specific reaction process into electrical signals through transducers, thereby quantifying the analytes. analyze. Compared with traditional detection techniques, it has the advantages of high sensitivity, high specificity, easy operation, fast analysis speed, low price, etc., and is easy to realize automatic operation. It has shown great promise in clinical diagnosis, medical care, environmental monitoring, food safety and other fields Good application prospects. However, the reported...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N27/60
Inventor 郑磊贾立永干宁王前李博巫远招
Owner SOUTHERN MEDICAL UNIVERSITY
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