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Microcystis toxin immune quantification test strip based on fluorescent microsphere mark

A technology of microcystin and fluorescent microspheres, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of wide range of microcystin pollution, detection methods that cannot meet the requirements, and narrow dynamic detection range. The effect of market demand, small error and high sensitivity

Inactive Publication Date: 2018-06-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This detection technology is easy to operate, does not require sample pretreatment, and has good specificity, but it uses a single reagent, which is difficult to control quality, and the sensitivity is not high, and the dynamic detection range is narrow, so it can only be used for qualitative analysis
[0005] The range of microcystin pollution in my country is wide, and the commonly used detection methods cannot meet the requirements. It is urgent to develop more sensitive and convenient methods that can be used for on-site rapid and high-throughput detection.

Method used

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  • Microcystis toxin immune quantification test strip based on fluorescent microsphere mark
  • Microcystis toxin immune quantification test strip based on fluorescent microsphere mark
  • Microcystis toxin immune quantification test strip based on fluorescent microsphere mark

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1 Fluorescent probe preparation of Eu-fluorescent microspheres labeled microcystin artificial antigen (or goat anti-rabbit secondary antibody)

[0036] The specific preparation method is as follows:

[0037] (1) Take 50 μL (1% solid content) of carboxyl fluorescent microspheres (purchased from Xiamen Nodao Technology Co., Ltd., particle size 300 nm) placed at 4°C, ultrasonically disperse, and add 600-800 μL of 2-(N-morphine at a concentration of 0.05M Phenyl)ethanesulfonic acid (MES, C 6 h 13 NO 4 S·H 2 O) Activation buffer, centrifuged at 16500rpm for 10-15min (the temperature is controlled at about 15°C during centrifugation);

[0038] (2) Discard the supernatant, add 600-800μL MES buffer, resuspend by ultrasonication, and repeat centrifugal washing 2-3 times;

[0039] (3) Discard the supernatant, add 200 μL MES buffer for ultrasonic resuspension, add 50 μL 10 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, C 8 h 17 N 3 HCl), 50...

Embodiment 2

[0045] Example 2 Assembly of Microcystin Immunity Test Strips Based on Labeled Fluorescent Microspheres

[0046] The structural diagram of the microcystin immune test strip is as follows: image 3 As shown, there are sample pads, nitrocellulose (NC) membrane and absorbent paper from left to right on the bottom plate. The key to test strip assembly is to ensure consistent transmissibility between each part. The sample pad is stacked on the NC. On the membrane, the two overlap by about 5mm. Similarly, the absorbent paper is stacked on the NC membrane, and the two overlap by about 5mm. Use a strip cutter to cut the pasted board into test strips about 4mm wide. Assemble and store sealed at 4°C for later use.

[0047] The assembly method is as follows: Spray goat anti-mouse secondary antibody (1mg / mL) on the nitrocellulose membrane as the detection line (T line), and spray rabbit IgG antibody (1mg / mL) on the nitrocellulose membrane as the quality control line (C line), the sprayi...

Embodiment 3

[0048] Example 3 Drawing of the Standard Curve Based on the Microcystin Immunization Test Strip of Labeled Fluorescent Microspheres

[0049] The method of drawing the standard curve is:

[0050] Eu-fluorescent microsphere-labeled microcystin artificial antigen (Eu-MC-LR-BSA) and goat anti-rabbit secondary antibody (Eu-Goat-anti-Rabbit) were used as fluorescent probes, and 30 μL of microcystin Standards, 30 μL of Eu-MC-LR-BSA, 30 μL of microcystin monoclonal antibody ascites and 5 μL of Eu-Goat-anti-Rabbit were added dropwise to a 96-well plate, shaken at room temperature for 10 minutes, and 70 μL of the mixture was slowly dropped into the test strip sample Pad, chromatographed at 37°C for 5min. Then record T value and C value by HG-98 immunological quantitative analyzer. The concentration gradient of microcystin standard substance is: 0μg / L, 0.1μg / L, 0.25μg / L, 0.5μg / L, 1μg / L, 2μg / L, 5μg / L. Take the logarithmic value of the standard substance concentration as the abscissa, a...

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Abstract

The invention discloses a microcystis toxin immune quantification test strip based on fluorescent microsphere mark and belongs to the technical field of immune analysis and quick detection. Accordingto the test strip, a fluorescent probe is prepared by marking fluorescent microspheres; the test strip is prepared from fluorescent microsphere-microcystis toxin artificial antigen and fluorescent microsphere-goat anti rabbit second antibody. The goat anti rabbit second antibody and rabbit IgG antibody are respectively sprayed on nitrocellulose membranes to serve as a detection line and a qualitycontrol line to prepare an immunochromatography test strip; competition immune method is utilized to quantitatively analyze microcystis toxin in a sample by reading a fluorescent value of the detection line on a fluorescence immunity analyzer. The method not only overcomes the defect that colloidal gold in a test strip technology is not easy to store; furthermore, a fluorescent probe preparation method has the advantages of simpleness and high efficiency.

Description

technical field [0001] The invention relates to a fluorescent microsphere-based microcystin immunoquantitative test strip, which belongs to the technical field of rapid detection of immune analysis. Background technique [0002] Microcystin (MC) is a class of heptapeptide monocyclic compound with biological activity, and it is a hepatotoxin with strong cancer-promoting effect. It is mainly produced by the freshwater algae Microcystis aeruginosa (Microcystisaeruginosa), and has considerable stability. So far, 80 different microcystin subtypes have been found, among which the variable amino acids are leucine (L) and arginine (R) microcystin-LR (Microcysitn-LR, MC-LR ) is the most toxic, currently known to be second only to dioxin in toxicity. A low dose of this toxin in drinking water can cause liver damage, promote the occurrence of liver cancer, and bring huge threats and hidden dangers to human health. [0003] At present, there are three main methods for detecting micro...

Claims

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Application Information

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IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 孙秀兰刘莹纪剑皮付伟张银志陆勇
Owner JIANGNAN UNIV
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