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Nanotechnology-based trace protein detection method

A protein detection and nanotechnology technology, applied in measurement devices, biological tests, material inspection products, etc., can solve problems such as poor sensitivity and linear range

Inactive Publication Date: 2011-01-12
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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Problems solved by technology

However, the currently used assay methods have the disadvantages of poor sensitivity and linear range.

Method used

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[0021] It is characterized in that the steps are: (1) using the double antibody sandwich method to capture PSA or other protein samples to be tested on the solid phase; (2) using tyramide signal amplification technology to generate a large amount of biotin deposition at the antigen-antibody binding site; (3) Aggregation of gold nanoparticles at biotin deposition sites and silver staining experiments; (4) Scanning and data analysis.

[0022] (1) Capture PSA or other protein samples to be tested by double-antibody sandwich method on the solid phase: use immunoenzyme-linked adsorption method to capture PSA and bind it to an antibody with horseradish peroxidase activity;

[0023] Dilute the PSA monoclonal antibody into a solution of a certain concentration, take 1 μL each and spot it on the epoxy-based substrate, place it in an incubator, vacuumize at 37°C overnight, take out the substrate, and add 10% BSA to the reaction pool The solution was blocked, and after blocking at room t...

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Abstract

The invention relates to a nanotechnology-based trace protein detection method, which combines enzyme-linked immunosorbent assay technology, tyramine signal amplification technology and the aggregation phenomenon of gold nanoparticles modified by different biological molecules, so an experimental method used for detecting trace proteins such as prostate specific antigen (PSA) and the like is established. The method comprises the following steps of: fixing an antibody aiming at the protein to be detected (such as the PSA) on the surface of a substrate; incubating another antibody with horseradish peroxidase (HRP) activity of the protein to be detected (such as the PSA) after capturing the protein to be detected in a sample, wherein the HRP catalyzes biotin-tyramide to generate biotin deposition under certain conditions; further amplifying a signal by using the aggregation phenomenon of the gold nanoparticles modified by biotin-labeled DNA and the gold nanoparticles modified by streptavidin; performing silver staining; and performing data analysis on an experimental result by using software. The method has the advantages of extremely low detection limit, wider detection range, capacity of detecting the antigen in a rabbit serum with complex compositions, and important application prospect.

Description

technical field [0001] The invention relates to a method for detecting trace proteins based on nanotechnology, which belongs to the technical field of analytical biochemistry. Background technique [0002] Measuring tiny amounts of proteins is essential in disease diagnosis and biomedical research. However, the currently used assay methods have the disadvantages of poor sensitivity and linear range. For example, prostate-specific antigen (PSA) is a single-chain glycoprotein encoded by hKLK3 gene, secreted by prostate epithelial cells, has chymotrypsin activity, and has a molecular weight of about 34000 g / mol. As a biomarker, PSA is widely used in disease screening, diagnosis and post-treatment monitoring. However, the content of PSA in human serum is extremely low. Therefore, higher requirements are placed on the detection limit and sensitivity of the detection method. [0003] Immunoenzyme-linked adsorbent assay is an immunoassay method widely used in clinical testing a...

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N21/33
Inventor 张玉祥沈海滢
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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