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Fluorescent microsphere immunochromatographic test strip for triple quantitative detection of fusarium toxin, preparation method and application of Fluorescent microsphere immunochromatographic test strip

A technology of immunochromatographic test strips and fluorescent microspheres, which is applied in measurement devices, analytical materials, instruments, etc., can solve the problems of single detection, limited sensitivity, instability of quantum dots, etc., and achieves high speed, improved sensitivity, coupled antibodies Simple and fast effects

Pending Publication Date: 2020-05-01
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the vast majority of commercial colloidal gold immunochromatographic test strips are single detection, and the sensitivity is limited.
Fluorescent microspheres are prepared by self-assembly of high-quality CdSe / ZnS quantum dots and biocompatible polymers. The fluorescence signal of quantum dots is amplified by coating nanospheres, which can significantly improve the sensitivity of immunodetection and also To solve the problem of quantum dot instability, the immunochromatographic test strips based on fluorescent microspheres for the simultaneous detection of three Fusarium toxins (FB1, DON and ZEN) have not been reported yet

Method used

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  • Fluorescent microsphere immunochromatographic test strip for triple quantitative detection of fusarium toxin, preparation method and application of Fluorescent microsphere immunochromatographic test strip
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Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1 Preparation of Triple Quantitative Detection Fusarium Toxin Fluorescent Immunochromatographic Test Strips

[0044] 1. Preparation of FB1, DON and ZEN monoclonal antibody-fluorescent microsphere markers. Take 10ug fluorescent microspheres, 0.5-1ug monoclonal antibody and a certain proportion of EDC (the mass ratio of fluorescent microspheres to EDC is 40:1) and add them to 400uL phosphate buffered saline solution (pH=6), then mix well Shake and incubate at 37°C for 10-20min, add 10% BSA to a final concentration of 0.1%, mix well and shake and incubate at 37°C for 10-20min; centrifuge at 12000rpm for 10min, discard the supernatant, and mix the FB1, DON and ZEN monoclonal antibodies - The fluorescent microsphere pellet was resuspended in a storage solution containing 0.9% sodium chloride, 1% BSA and 0.09% sodium azide, and stored at 4°C after ultrasonication for 20s.

[0045] 2. Spray the gold-labeled antibody onto the gold-labeled pad. Before coating the gol...

Embodiment 2

[0049] Embodiment 2 establishes standard curve

[0050] Prepare a range of concentrations (0 / 0 / 0, 0.47 / 0.195 / 0.094, 0.94 / 0.39 / 0.188, 1.875 / 0.78 / 0.375, 3.75 / 1.56 / 0.75, 7.5 / 3.12 / 1.25, 15 / 6.25 / 2.5, 30 / 12.5 / 5, 60 / 25 / 10, 120 / 50 / 20, 240 / 100 / 40ng / mL) FB1, DON and ZEN standards, the prepared solution is containing 5% methanol, 0.5% Tween20, 0.5% PvP k30 10 mM PBS solution. Get 100 μ L of standard solution and drop on the test strip sample pad prepared in embodiment 1, measure the fluorescence value on each detection line and control line on the test strip after 25min, obtain the detection line and the fluorescence intensity ratio on the control line (F=T / C), then take the natural logarithm (LogC) of the concentration value of the standard series as the abscissa and the fluorescence intensity value percentage (F / F o ) to establish a corresponding standard curve for the ordinate, such as figure 2 shown, where F o It is the T / C value when the standard substance concentration is ...

Embodiment 3

[0051] Example 3 sample detection and result analysis

[0052] Add 5 g of sample to 20 mL of extract (methanol: water = 60: 40, v / v), shake vigorously in a shaker for 30 min. After standing still for 30 min, the supernatant was centrifuged at 6000 rpm for 10 min, then the supernatant was filtered with a 0.22 μm polytetrafluoroethylene filter, and diluted 15 times with 10 mM PBS containing 0.5% PvPk30 and 0.5% Tween20, and finally the diluted The sample was centrifuged again at 12000rpm for 10min, and the supernatant was collected. Take 100 μL of the treated sample solution and drop it on the sample pad of the test strip, measure the fluorescence value on the test line and the control line on the test strip after 25 minutes, calculate the content of each toxin in the sample according to the corresponding fluorescence value and the standard curve, like figure 2 shown.

[0053] The results showed that the IC50 values ​​of the above test strips for simultaneous detection of ...

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Abstract

The invention discloses a fluorescent microsphere immunochromatographic test strip for triple quantitative detection of fusarium toxin, and a preparation method and application of the fluorescent microsphere immunochromatographic test strip. The method comprises the following steps of sequentially sticking a nitrocellulose membrane, a binding pad, a sample pad and a water-absorbing pad on a base plate, wherein the nitrocellulose membrane is respectively coated with detection lines of ZEN-BSA, DON-BSA and FB1-BSA and a control line of goat anti-mouse IgG antibody, and the binding pad is sprayedwith the monoclonal antibody-fluorescent microsphere marker mixture of FB1, DON and ZEN, and assembling, cutting strips to obtain the test strips. The detection of three kinds of fusarium toxins is integrated into one test strip, the formed multi-channel detection efficiency is high, the obtained test strip is good in specificity, high in sensitivity, simple in operation and capable of being usedby non-professionals, meets the requirements of quickly and accurately judging the specific contents of FB1, DON and ZEN toxins in grain storage and sale organizations, entry and exit quarantine, customs, production enterprises, supervision departments and the like, and is convenient for basic popularization and application.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and is an immunochromatographic test that uses fluorescent microspheres combined with quantum dots as a signal amplification material and simultaneously detects fumonisin B1, deoxynivalenol and zearalenone. Paper strip, its preparation method and application. Background technique [0002] Mycotoxins are secondary metabolites produced during the growth of fungi, which are harmful to humans and animals. Common mycotoxins are mainly derived from Aspergillus, Penicillium and Fusarium, and more than 400 mycotoxins have been discovered so far. Fusarium can produce a variety of toxins during the growth process, among which fumonisins B1 (FB1), deoxynivalenol (DON) and zearalenone (Zearalenone, ZEN) belong to Common Fusarium toxins are widely distributed in nature and mainly contaminate various crops such as corn, wheat, barley and oats and their products. Different Fusarium species have ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/558G01N33/577G01N33/543
CPCG01N33/533G01N33/54313G01N33/558G01N33/577
Inventor 严亚贤侯思露孙建和
Owner SHANGHAI JIAO TONG UNIV
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