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Indirect background fluorescence colloidal gold immunochromatographic test strip based on double-labeled signal amplification and application thereof

A colloidal gold, double-labeled technology, applied in the fields of food safety detection and biomedicine, can solve the problems of matrix interference, sensitivity needs to be improved, repeatability is not optimistic, etc.

Inactive Publication Date: 2020-03-10
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the immunochromatography technology based on colloidal gold as a marker is the most successful and widely used rapid screening method. However, due to the serious matrix interference in samples from different regions, varieties, and times, the accuracy of the test strips cannot be detected. The accuracy and repeatability are not optimistic; and the stability and sensitivity of test strip detection results still need to be improved

Method used

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  • Indirect background fluorescence colloidal gold immunochromatographic test strip based on double-labeled signal amplification and application thereof
  • Indirect background fluorescence colloidal gold immunochromatographic test strip based on double-labeled signal amplification and application thereof
  • Indirect background fluorescence colloidal gold immunochromatographic test strip based on double-labeled signal amplification and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1: Preparation of immunomagnetic microspheres and its application in enriching clenbuterol hydrochloride in swine urine samples

[0091] 1. Preparation of immunomagnetic microspheres

[0092] 1. Preparation of Suspension A

[0093] Weigh 0.1g Fe 3 o 4 Add 20 mL of distilled water to the magnetic microspheres, and form suspension A after ultrasonication for 30 min.

[0094] 2. Preparation of Suspension B

[0095] 0.8g chitosan (purchased from Bailingwei Technology Co., Ltd., degree of deacetylation: 95%) was dissolved in 15mL of dilute acetic acid solution with a volume fraction of 2%, to obtain a homogeneous suspension B with a mass fraction of 2%.

[0096] 3. Fe 3 o 4 @Preparation of chitosan magnetic microspheres

[0097] Mix suspension A and suspension B according to the volume ratio of 4:1, stir for 1 h, spray dry by spray dryer for 24 h, and then calcinate at 800 °C for 4 h under nitrogen to obtain Fe 3 o 4 @Chitosan magnetic microspheres.

[0098...

Embodiment 2

[0110] Example 2: Preparation of double-labeled indirect background fluorescent colloidal gold test strips

[0111] 1. Preparation of double-labeled colloidal gold

[0112] 1. Preparation of colloidal gold-clenbuterol hydrochloride monoclonal antibody-biotin (GNPs-clenbuterol hydrochloride antibody-biotin)

[0113] (1) Preparation of biotinylated clenbuterol monoclonal antibody solution

[0114] Aminated biotin (purchased from SIGMA Company) with a concentration of 3.8 mg / ml was added to 1 mg of clenbuterol hydrochloride monoclonal antibody, and after rotating for 1 hour, dialyzed three times with a dialysis membrane (8k MWCO), buffer (pH7 .4) The formula is as follows: 50mM PB, 75mM NaCl, each time 2L of dialysis for more than 3h. Then use a pipette to carefully suck out the biotinylated antibody from the dialysis bag, adjust the concentration of the biotinylated antibody with a 50 mM PBS solution, and obtain a biotinylated clenbuterol monoclonal antibody with a concentrati...

Embodiment 3

[0129] Embodiment 3: Quantitative and qualitative detection of clenbuterol hydrochloride

[0130] 1. Qualitative detection method of clenbuterol hydrochloride

[0131] 1. Enrich and purify the sample to be tested using the immunomagnetic microspheres in Example 1 to obtain a processed sample.

[0132] 2. Combine 150 μL of the treated sample with 3 μL of the GNPs-clenbuterol hydrochloride antibody-biotin solution prepared in step 1 of Example 2 and 3 μL of GNPs-streptavidin prepared in step 1 of Example 2 The solution was mixed and incubated at room temperature for 3 min to obtain a mixed solution.

[0133] 3. Take 120 μL of the mixed solution and drop it on the sample pad of the test strip. Qualitative observation with naked eyes after 10 min.

[0134] If both the C line and the T line are red, the sample to be tested does not contain clenbuterol hydrochloride;

[0135] If the C line is red and the T line is not, the sample to be tested contains clenbuterol hydrochloride. ...

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Abstract

The invention discloses an indirect background fluorescence colloidal gold immunochromatographic test strip based on double-labeled signal amplification and application thereof. The test strip comprises five parts, namely a PVC plastic back plate paved with fluorescence, a PVC bottom plate, a nitrocellulose membrane, a sample pad and absorbent paper. The invention also discloses a double-labeled colloidal gold probe, the double-labeled colloidal gold probe is composed of a gold nanoparticle labeled biotinylated anti-target object antibody and gold nanoparticle labeled streptavidin, and signalamplification is realized by using superstrong affinity between biotin-streptavidin molecules. According to the fluorescent colloidal gold immunochromatography test strip disclosed by the invention, asample is pretreated by utilizing the immunomagnetic microspheres before detection, and an enriched target object can be efficiently and rapidly separated in an external magnetic field. The test strip has the advantages of good stability, high sensitivity and no matrix interference, can realize rapid qualitative or quantitative detection of the sample, is simple in operation steps, strong in controllability, and has good application prospects.

Description

technical field [0001] The invention belongs to the technical fields of food safety detection and biomedicine, and in particular relates to an indirect background fluorescent colloidal gold immunochromatographic test strip based on double-label signal amplification and an application thereof. Background technique [0002] With my country's accession to the WTO, people's demand for food has gradually changed to quality. The problem of residues of harmful substances has become an important indicator of food safety testing in all countries in the world. Although the Chinese government has also formulated a series of laws, regulations and technical standards to limit the residues of harmful substances, it has strengthened residue detection, established a residue detection system, and provided safety and hygiene to the society. It is imperative to protect human health. [0003] At present, the detection of harmful substance residues mainly uses instruments and rapid detection te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/558
CPCG01N33/533G01N33/558
Inventor 江海洋沈建忠郑丕苗吴聪明丁双阳王战辉柯跃斌温凯曾于洋梁德媚
Owner CHINA AGRI UNIV
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