Immunosensor for detecting aspergillus parasiticus used for producing aflatoxin and preparing method thereof

A technology of aflatoxin and parasitic Aspergillus, applied in the direction of instruments, measuring devices, scientific instruments, etc., can solve the problems of cumbersome detection steps, long detection time, reagent human body and environmental hazards, and achieve the promotion of electron transfer and short response time , cheap effect

Active Publication Date: 2014-05-14
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The identification medium method is convenient to obtain materials, but the sample pretreatment is complicated, the operation process is cumbersome, the detection cycle is long, the sensitivity is low, and it is time-consuming and laborious.
ELISA requires special equipment, the detection steps are cumbersome, expensive, long detection time, and prone to false positives
Although the development of molecular-level detection methods of multiplex PCR and real-time fluorescence quantitative PCR has advantages over traditional methods, the equipment required is expensive, the detection process is complicated, the cost is high, and the detection environment and operators have high requirements for professional skills. The reagents used are very harmful to the human body and the environment, and the specificity of this method is not good due to the complexity of the toxin-producing genes

Method used

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  • Immunosensor for detecting aspergillus parasiticus used for producing aflatoxin and preparing method thereof
  • Immunosensor for detecting aspergillus parasiticus used for producing aflatoxin and preparing method thereof
  • Immunosensor for detecting aspergillus parasiticus used for producing aflatoxin and preparing method thereof

Examples

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Effect test

Embodiment 1

[0033] Step 1, the construction of the immunosensor: After polishing and cleaning the bare gold electrode, firstly immerse it in 0.3%-0.7% (m / v) L-cysteine, and soak it at 35-40°C for 3-7h. Then, stand overnight at 4°C to deposit gold nanoparticles, take it out, wash with ultrapure water, and blow dry. Apply 3-15 μL of homemade polyclonal antibody on the surface of the electrode, and incubate at 37°C for 1 hour. Finally, 10 μL of 5% skim milk-PBS solution was added dropwise as a masking reagent to block the non-specific adsorption sites, reacted at 37°C and washed with ultrapure water. Electrodes that are not used immediately need to be dried with nitrogen and stored in PBS with a pH of 7.4 at 4°C until use.

[0034] Step 2: Immunosensor detection of Aspergillus parasitica spore liquid: Aspergillus parasitica was cultured at 28° C. on a PDA slant solid medium for 5-7 days, and the spores were eluted with 10 mL of mold distilled water containing 0.05% Tween to prepare spores ...

Embodiment 2

[0036] Determination of spiked Aspergillus parasitica in actual samples of naturally fermented soybean paste

[0037] Step 1, the processing of naturally fermented soybean paste samples: the soybean paste sample is ground and sterilized, take 2g of soybean paste under aseptic operation, dilute it with 1:1000 sterile ultrapure water, and add the Aspergillus parasitica spore standard product, so that the final concentration is 150-1500cfu / mL. And configure a blank control of soybean paste without Aspergillus parasiticus spores.

[0038]Step 2, construction of the immunosensor: after polishing and cleaning the bare gold electrode, firstly immerse it in 0.5% (m / v) L-cysteine, and soak it at 37° C. for 5 hours. Then, stand overnight at 4°C to deposit gold nanoparticles, take it out, wash with ultrapure water, and blow dry. Apply 10 μL of homemade polyclonal antibody on the surface of the electrode, and incubate at 37°C for 1 hour. Finally, 10 μL of 5% skim milk-PBS solution was ...

Embodiment 3

[0043] Determination of Aspergillus parasitica in corn flour samples

[0044] Step 1, processing of corn flour samples: take 2 g of corn flour under aseptic operation, and grind them appropriately. Dilute with a certain concentration of sterilized ultrapure water after grinding.

[0045] Step 2, construction of the immunosensor: after polishing and cleaning the bare gold electrode, firstly immerse it in 0.5% (m / v) L-cysteine, and soak it at 37° C. for 5 hours. Then, stand overnight at 4°C to deposit gold nanoparticles, take it out, wash with ultrapure water, and blow dry. Apply 10 μL polyclonal antibody to aflatoxin-producing Aspergillus on the surface of the electrode, and incubate at 37°C for 1 hour. Finally, 10 μL of 5% skim milk-PBS solution was added dropwise as a masking reagent to block the non-specific adsorption sites, reacted at 37°C and washed with ultrapure water. Electrodes that are not used immediately need to be dried with nitrogen and stored in PBS with a pH...

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Abstract

The invention discloses an immunosensor for detecting aspergillus parasiticus used for producing aflatoxin and a preparing method thereof. The immunosensor is obtained by the following step that: nano-Au, L- cysteine and aspergillus parasiticus polyoxin are self-assembled and modified on a gold electrode. According to the invention, the chemical amplification of the nano-Au and the specificity of the immunosensor are combined, and the advantages of the nano-Au and the immunosensor are also merged, so that the immunosensor simultaneously has the specificity of immune reaction and the quickness and sensitivity of electrochemistry analysis, and can accurately detect the low-content aspergillus parasiticus used for producing the aflatoxin.

Description

technical field [0001] The invention belongs to the technical field of rapid detection of pathogenic fungi, in particular to a label-free immune biosensor for detecting aflatoxin-producing aspergillus based on the reaction of antigens and antibodies and a preparation method thereof. Background technique [0002] Aflatoxin is a highly toxic and strong carcinogen. It is the most stable of all mycotoxins found so far. It was designated as a Class I carcinogen in 1988. 68 times. Aflatoxins are widely distributed, and almost all grains, feedstuffs and various foods (including animal products) can be used as growth substrates for aflatoxin-producing bacteria, seriously endangering human health. [0003] To prevent the harm of aflatoxin, the first condition is to be able to quickly and accurately detect whether grains, food, etc. are contaminated by aflatoxin-producing Aspergillus. At present, the detection methods of aflatoxin-producing Aspergillus mainly include traditional dif...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N27/26G01N27/327
Inventor 孙秀兰晏丽吴龙云张银志王淼赵建新
Owner JIANGNAN UNIV
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