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Visual immunoassay method for detecting aspergillus flavus M1

A flavus, fluorescence detection technology, applied in the direction of analysis materials, measurement devices, biological testing, etc., can solve the problems of lack of signal transmission mechanism, increase the total time of detection, detection complexity, lack of specificity, etc., to improve practicability and detection efficiency, shortening the total detection time, and good economic value

Inactive Publication Date: 2021-01-15
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It mainly adsorbs the target compound through π-π stacking force, electrostatic adsorption, hydrogen bond and other forces, lacking specificity
In addition, GMC is often only used for the enrichment and purification of samples, and lacks a signal transmission mechanism, which separates the enrichment and purification of samples from the acquisition of detection signals in time and space, increasing the total time and complexity of detection.

Method used

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  • Visual immunoassay method for detecting aspergillus flavus M1
  • Visual immunoassay method for detecting aspergillus flavus M1
  • Visual immunoassay method for detecting aspergillus flavus M1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The synthesis of embodiment 1 magnetic graphene

[0024] Accurately weigh FeCl 3 0.144g and FeCl 2 0.056g solution 2.22mL deionized water; Accurately weigh 15mg of graphene oxide and disperse it into 10mL deionized water under ultrasonic conditions. FeCl 3 and FeCl 2 The mixed solution was slowly dropped into the graphene oxide. After that, 100 mg of L-cysteine ​​was accurately weighed, and 0.4 mL of 25% ammonia solution was added to the above reaction solution, ultrasonically dispersed for 5 min, and the reaction solution was placed in a water bath at 90° C. for static reaction for 10 h. After that, centrifuge at 10000g to obtain magnetic graphene. Characterized by transmission electron microscopy, high-resolution transmission electron microscopy, energy dispersive X-ray, magnetic properties and ultraviolet absorption spectra, the results are shown in figure 1 , Fe 3 o 4 Evenly distributed on the graphene sheet, the nanometer diameter is 5.2nm. Energy dispers...

Embodiment 2

[0025] Embodiment 2 The coupling of magnetic graphene and antibody

[0026] 2.1 Solution preparation

[0027] Morpholineethanesulfonic acid buffer (0.05M MES): Accurately weigh 9.7g of morpholineethanesulfonic acid, and dilute to 1000mL.

[0028] Phosphate buffer (0.01MPBS, pH 7.4): Accurately weigh 8.00g of NaCl, KH 2 PO 4 0.20g, Na 2 HPO 4 212H 2 O2.90g, KCl 0.20g, dissolved in a small amount of ultrapure water, and set the volume to 1000mL.

[0029] Washing solution (0.01M PBST, pH 7.4): Accurately weigh 8.00g of NaCl, KH 2 PO 4 0.20g, Na 2 HPO 4 212H 2O2.90g, KCl 0.20g, dissolve in a small amount of ultrapure water, add 0.50mL Tween 20, and dilute to 1000mL.

[0030] Blocking solution: Accurately weigh 10.00 g of bovine serum albumin (BSA), add 100 mL of deionized water, and stir until the protein is completely dissolved.

[0031] 2.2 Coupling of magnetic graphene and antibody

[0032] Accurately weigh 2mg of the magnetic graphene prepared above, add 0.8mL 0...

Embodiment 3

[0034] Synthesis of embodiment 3 fluorescent probes

[0035] Weigh AFB 1 4 mg, 3.8 mg of oxycarboxymethylhydroxylamine hemihydrochloride (CMO), dissolved in 5 mL of pyridine solution, then transferred to a round-bottomed flask with a condensing device, 70 ° C magnetic stirring reaction for 6 h, the reaction solution in a water bath nitrogen instrument Blow dry (70°C) to obtain AFB 1 -CMO.

[0036] Take AFB 1 -CMO2 mg, dissolved in 0.5mL N,N-dimethylformamide (DMF) solution, added 6mg EDC and 2mgNHS, stirred at room temperature for 12h, centrifuged at 3,000rmp / min for 5min, and the supernatant was the activated hapten solution.

[0037] EDF was obtained by reacting fluorescein isothiocyanate (FITC) with ethylenediamine: take 100mg of ethylenediamine, add 50mL of methanol, drop in 200μL of triethylamine, and magnetically stir to fully dissolve it; take 60mg of FITC, dissolve it in 10mL of methanol , drop 100 μL of triethylamine, and magnetically stir to fully dissolve it; ...

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Abstract

The invention belongs to the technical field of immunoassay, and particularly relates to a visual immunoassay method based on magnetic graphene, which is used for detecting aspergillus flavus M1 in milk. The preparation method comprises the following steps: synthesizing magnetic graphene by adopting a one-pot method; coupling the magnetic graphene with a monoclonal antibody of aspergillus flavus M1 by adopting a chemical method, and closing bovine serum albumin; preparing a fluorescent probe aspergillus flavus B1-EDF; adopting a direct competition method, wherein a target compound aspergillusflavus M1 and a fluorescent probe aspergillus flavus B1-EDF compete for an aspergillus flavus M1 monoclonal antibody on magnetic graphene at the same time, and performing qualitative analysis under anultraviolet lamp according to a fluorescence signal of a graphene quenching fluorescent probe; and calculating the content of the target compound aspergillus flavus M1 according to the quantity of fluorescence signals of the graphene quenching fluorescence probe, and carrying out quantitative analysis. Compared with the prior art, the visual immunoassay method provided by the invention can integrate sample enrichment and purification and visual detection, and has the advantages of simplicity, convenience, rapidness, sensitivity, accuracy and the like.

Description

technical field [0001] The invention belongs to the technical fields of residue analysis and immunology of chemical hazard factors. It specifically relates to a magnetic graphene-based visual immunoassay method using Aspergillus flavus M 1 As a detection target, detection of Aspergillus flavus M in food 1 . Background technique [0002] Fluorescence resonance energy transfer (FRET) refers to the non-radiative energy transfer between a donor and an acceptor that is close enough (about 10nm), and is often used as a homogeneous immunoassay for chemical hazards. Detection has the advantages of fast analysis and no need for cumbersome washing steps. However, common FRET often selects fluorescent groups with smaller Snooker shifts as energy donors or acceptors, which makes spectral resolution difficult and the energy transfer efficiency is low. In recent literature reports, it was found that reduced graphene oxide (rGO) has a super-strong fluorescence quenching performance, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58G01N33/533G01N33/577G01N33/543
CPCG01N33/533G01N33/54326G01N33/54373G01N33/577G01N33/582
Inventor 张西亚毛烨炫黄现青宋莲军乔明武李倩刘亮赵秋艳李家寅丁明月
Owner HENAN AGRICULTURAL UNIVERSITY
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