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In-vitro sandwich immunoassay process with magnetic separation for detecting alpha fetoprotein

A technology of alpha-fetoprotein and sandwich immunity, applied in the field of detection and analysis of alpha-fetoprotein, can solve the problems of signal instability, poor sensor reproducibility, and long time consumption

Inactive Publication Date: 2013-01-16
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The problem to be solved by the present invention is that the traditional construction of immunosensors is generally a process of layer-by-layer assembly, which takes a long time and is easily disturbed by external conditions during the construction process, resulting in poor reproducibility of the sensor when detecting a certain target substance. unstable signal

Method used

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  • In-vitro sandwich immunoassay process with magnetic separation for detecting alpha fetoprotein
  • In-vitro sandwich immunoassay process with magnetic separation for detecting alpha fetoprotein
  • In-vitro sandwich immunoassay process with magnetic separation for detecting alpha fetoprotein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of in vitro sandwich immunosensor based on magnetic separation

[0038] The preparation of an immunosensor for separation and enrichment and for the detection of alpha-fetoprotein antigen is realized based on an immune sandwich complex modified electrode formed by a magnetic capture probe constructed of a magnetic nanomaterial and a quantum dot-labeled signal probe.

[0039] Its preparation method is as follows:

[0040] (1) Preparation of magnetic capture probe: Weigh 20 mg of magnetic nano-Fe 3 o 4 -Ultrasonic dispersion of Au for 15-20min in PBS buffer solution with pH 7.4 to prepare uniformly dispersed 1mg mL -1 Fe 3 o 4 - Au / PBS dispersion. Draw 1mL and 50μg mL -1 The alpha-fetoprotein antibody (Ab1) was mixed and stirred at 4°C for 24 hours, and 1 mL of 1% bovine serum albumin (BSA) was added to block the active sites of other unbound antibodies. Magnetic separation and washing without magnetic nano-Fe 3 o 4 - Au-immobilized Abl, was...

Embodiment 2

[0045] Example 2 Comparison of Electrochemiluminescence Responses of Several Different Modified Electrodes

[0046] A comparative study of the electrochemiluminescence response signals of different modified electrodes, the results are as follows: figure 1 . The bare electrode (curve a) contains 0.1mol / L PBS (pH=7.4) and 0.1mol / L KCl in 0.1mol / L KCl 2 S 2 o 8 In solution, its electrochemiluminescence response signal is very weak; the electrode surface is modified with Fe 3 o 4 After -Au / Ab1 / AFP (curve b), the electrochemiluminescent signal is also very weak, because there is no luminescent signal source substance on the electrode surface and the modified complex, so the electroluminescent signal is very weak and is a background signal ; When the electrode surface is modified with Fe 3 o 4 -Au / Ab1 / AFP / Ab2 / CdS (curve c) has a significantly increased electrochemiluminescence signal, which is a sandwich immune complex with a signal source substance modified to the electrode ...

Embodiment 3

[0047] Example 3 Establishment of a detection method for alpha-fetoprotein antigen by in vitro sandwich immunization based on magnetic separation

[0048] Prepare a standard alpha-fetoprotein antigen solution with a concentration distribution of 0 to 5.0 ng / mL; first, the polished bare electrode contains 0.1 mol / L PBS (pH=7.4) and 0.1 mol / L KCl in 0.1 mol / L KCl 2 S 2 o 8 Scan the background signal in the solution and record the signal intensity I 0 Then according to the above-mentioned establishment of standard curve steps to measure the corresponding electrochemiluminescence signal intensity value of different concentrations of alpha-fetoprotein antigen, its scanning solution is all the same that is: 0.1mol / L PBS (pH=7.4 is contained in the KCl of 0.1mol / L ) and 0.1mol / L K 2 S 2 o 8 Solution, the electrochemiluminescent signal intensity corresponding to different alpha-fetoprotein antigens was recorded as I 1 , I 2 , I 3 …,Such as figure 2 shown. The above experime...

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Abstract

The invention relates to the field of analysis and testing, and an in-vitro sandwich immunoassay process with magnetic separation for detecting alpha fetoprotein is built. The in-vitro sandwich immunoassay process is that a sandwich immune compound with a magnetic separation characteristic is obtained by catching an alpha fetoprotein antigen and a signal probe sandwich fixed by a quantum dot through a magnetic probe. The in-vitro sandwich immunoassay process for detecting the alpha fetoprotein builds an immunosensor to implement the detection of a target substance. The built immunosensor comprises a substrate electrode and modifies a prepared magnetic sandwich immune compound on the surface of the substrate electrode; and the relative electrogenerated chemiluminescent signals are different due to the different quantities of quantum dots relative to the alpha fetoprotein antigens with the different concentrations, so that the quantitative analysis of the alpha fetoprotein can be implemented. The in-vitro sandwich immunoassay process based on magnetism provided by the invention is good in anti-interference performance; and the detection method of the alpha fetoprotein is easy, the sample preprocessing is simple, and the detection and the analysis of content of the alpha fetoprotein in a serum sample with high flexibility can be adapted.

Description

technical field [0001] The present invention relates to the detection and analysis of alpha-fetoprotein, which is based on an in vitro sandwich immunoassay method combined with magnetic nanoparticle separation technology for quantitative analysis by electrochemiluminescence analysis method to construct a high-sensitivity, high-selectivity magnetic separation electrolysis method. A chemiluminescent immunosensor for the detection of alpha-fetoprotein. Background technique [0002] Alpha-fetoprotein is a specific marker of liver cancer. To diagnose primary liver cancer, detecting the content of alpha-fetoprotein is one of the important means. High levels of alpha-fetoprotein in normal people may be associated with chronic hepatitis, congenital biliary occlusion, malformed fetuses, and germ cell tumors. Therefore, early screening and early diagnosis are very necessary to prevent possible negative effects . [0003] At present, the detection of alpha-fetoprotein mainly includes...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/76
Inventor 干宁周汉坤李天华曾少林周靖熊萍杜晓雯
Owner NINGBO UNIV
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