Magnetic fluorescent microsphere immunochromatography quantitative detection method

Abstract

The invention discloses a magnetic fluorescent microsphere immunochromatography quantitative detection method. In the method, respective excellent characteristics of magnetic nano particles and quantum dots are fully utilized, and an immunochromatography technology is combined to realize fluorescent quantitative detection on the basis of optimizing the structure and ingredients of a test strip. The method has a function of amplifying signals; and compared with the conventional colloidal gold immunochromatography method, the method has the advantages of high mark stability, low non-specificity, high sensitivity, wide linear range and accurate quantification. The invention provides a simple, accurate, specific and cheap detection tool for blood samples, urine samples, spittle, excrement and the like, so the method can be widely applied to the fields of medical technology, food safety, veterinary drug residues, environmental monitoring, drug detection and the like.

Description

technical field

[0001] The present invention relates to a method based on immunochromatography technology combined with magnetic separation technology and fluorescence amplification detection technology, in particular to a quantitative detection method of magnetic fluorescent microsphere immunochromatography, which is effective for pathogens, major diseases (such as cardiovascular disease, It belongs to the field of fluorescence detection technology for qualitative and quantitative detection of target analytes such as tumors), drugs, drug residues and food safety. Background technique

[0002] Immunochromatography technology originated in the 1990s, and has attracted widespread attention because of its simplicity and speed. In the past ten years, people have continuously researched and improved this technology, and many achievements have been made. In particular, colloidal gold immunochromatography has been widely used in clinical testing. However, the preparation system o...

Examples

Embodiment 1

[0059] Example 1: Double-antibody sandwich determination of human chorionic gonadotropin (hCG)

[0060] (1) Synthesis and functionalization of magnetic nanoparticles

[0061] The preparation of magnetic nanoparticles is mainly divided into physical methods and chemical methods, among which chemical methods are more commonly used, and can be mainly divided into co-precipitation method, pyrolysis method, microemulsion method, hydrothermal method and sonochemical method. The co-precipitation method is selected in the embodiment of the present invention, which has the advantages of simple equipment, convenient operation, and low cost, and is a traditional method for preparing magnetic nanoparticles. The specific operation steps are as follows:

[0062] 1) Divalent iron salt (FeCl 2 4H 2 O) and ferric salt (FeCl 3 ·6H 2 O) The solution is ultrasonically mixed according to a mol ratio of 2:1, and added to a three-necked round-bottomed flask, and the precipitant NH in the droppi...

Embodiment 2

[0083] Embodiment 2: Competitive method is determined organophosphorus

[0084] (1) Synthesis of magnetic nanoparticles

[0085] Prepare Fe according to the method of embodiment 1 3 o 4 magnetic nanoparticles.

[0086] (2) Acidification of carboxymethyl chitosan

[0087] Take carboxymethyl chitosan sodium salt and add 1.5M HCl dropwise to dissolve, acidify for 30min, then add dropwise into absolute ethanol, let it stand for 12h to precipitate carboxymethyl chitosan, centrifuge at 15000rpm for 10min, and repeat with absolute ethanol for more times of washing to obtain carboxymethyl chitosan CMCH.

[0088] (3) Preparation of antibody-modified magnetic-fluorescence composite microspheres, the schematic diagram of which is constructed as follows image 3 shown

[0089] Fe 3 o 4 Magnetic nanoparticles were mixed with 1 mL of carboxymethyl chitosan solution, and EDC was used as a cross-linking agent, ultrasonically reacted for 1 h, magnetically separated, washed with ethanol...

technical field

[0001] The present invention relates to a method based on immunochromatography technology combined with magnetic separation technology and fluorescence amplification detection technology, in particular to a quantitative detection method of magnetic fluorescent microsphere immunochromatography, which is effective for pathogens, major diseases (such as cardiovascular disease, It belongs to the field of fluorescence detection technology for qualitative and quantitative detection of target analytes such as tumors), drugs, drug residues and food safety. Background technique

[0002] Immunochromatography technology originated in the 1990s, and has attracted widespread attention because of its simplicity and speed. In the past ten years, people have continuously researched and improved this technology, and many achievements have been made. In particular, colloidal gold immunochromatography has been widely used in clinical testing. However, the preparation system o...

Examples

Embodiment 1

[0059] Example 1: Double-antibody sandwich determination of human chorionic gonadotropin (hCG)

[0060] (1) Synthesis and functionalization of magnetic nanoparticles

[0061] The preparation of magnetic nanoparticles is mainly divided into physical methods and chemical methods, among which chemical methods are more commonly used, and can be mainly divided into co-precipitation method, pyrolysis method, microemulsion method, hydrothermal method and sonochemical method. The co-precipitation method is selected in the embodiment of the present invention, which has the advantages of simple equipment, convenient operation, and low cost, and is a traditional method for preparing magnetic nanoparticles. The specific operation steps are as follows:

[0062] 1) Divalent iron salt (FeCl 2 4H 2 O) and ferric salt (FeCl 3 ·6H 2 O) The solution is ultrasonically mixed according to a mol ratio of 2:1, and added to a three-necked round-bottomed flask, and the precipitant NH in the droppi...

Embodiment 2

[0083] Embodiment 2: Competitive method is determined organophosphorus

[0084] (1) Synthesis of magnetic nanoparticles

[0085] Prepare Fe according to the method of embodiment 1 3 o 4 magnetic nanoparticles.

[0086] (2) Acidification of carboxymethyl chitosan

[0087] Take carboxymethyl chitosan sodium salt and add 1.5M HCl dropwise to dissolve, acidify for 30min, then add dropwise into absolute ethanol, let it stand for 12h to precipitate carboxymethyl chitosan, centrifuge at 15000rpm for 10min, and repeat with absolute ethanol for more times of washing to obtain carboxymethyl chitosan CMCH.

[0088] (3) Preparation of antibody-modified magnetic-fluorescence composite microspheres, the schematic diagram of which is constructed as follows image 3 shown

[0089] Fe 3 o 4 Magnetic nanoparticles were mixed with 1 mL of carboxymethyl chitosan solution, and EDC was used as a cross-linking agent, ultrasonically reacted for 1 h, magnetically separated, washed with ethanol...

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