Ratio fluorescence biosensor based on gold nanoclusters, preparation method of ratio fluorescence biosensor and application of ratio fluorescence biosensor in detection of aspergillus mould

A biosensor, ratiometric fluorescence technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc. Mild conditions, portable operation, easy and fast operation

Pending Publication Date: 2022-07-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, PCR detection can not achieve quantitative, can only stay in the qualitative stage
At the same time, t

Method used

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  • Ratio fluorescence biosensor based on gold nanoclusters, preparation method of ratio fluorescence biosensor and application of ratio fluorescence biosensor in detection of aspergillus mould
  • Ratio fluorescence biosensor based on gold nanoclusters, preparation method of ratio fluorescence biosensor and application of ratio fluorescence biosensor in detection of aspergillus mould
  • Ratio fluorescence biosensor based on gold nanoclusters, preparation method of ratio fluorescence biosensor and application of ratio fluorescence biosensor in detection of aspergillus mould

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1F

[0063] Example 1 Synthesis and characterization of FITC@AuNCs

[0064] Gold nanoclusters (AuNCs): 10 mL of HAuCl at 37 °C 4 (10 mmol / L) was added to 10 mL of BSA solution (50 mg mL -1 ), and then 1 mL of NaOH solution (1 mol / L) was added. After the reaction was continued for 12 h under vigorous stirring, it was dialyzed in ultrapure water for 24 h (water was changed every 8 h), and the prepared AuNCs were stored at 4 °C for use. For material characterization and subsequent synthesis, a freeze dryer was used for drying, and the solid AuNCs were redispersed in PBS buffer (pH=7.4) and stored at 4°C for later use.

[0065] FITC@AuNCs: Add 1 mg / mL ethanol solution of FITC to AuNCs reconstituted in PBS buffer (pH=7.4), stir in dark at room temperature for 12 h, and then add 0.05 mol / L PBS buffer (pH=7.4) ) was dialyzed for 8 h to obtain the product FITC@AuNCs, which were stored in a refrigerator at 4 °C.

[0066] The successful connection of FITC and AuNCs was verified by charac...

Embodiment 2

[0071] Design and verification of primers in Example 2

[0072] (1) Extracting the genomic DNA of the tested strain by the kit method

[0073] Take the activated Aspergillus niger, Aspergillus aegypti, Penicillium viridis and Fusarium oxysporum in potato liquid medium at 25℃, 180r / min shaking culture for 40h, Aspergillus flavus in wort liquid medium at 28℃, 180r / min After shaking culture for 40 hours, the mycelial balls in the liquid medium were taken to extract the fungal genome through the kit (the kit here is the Biospin Fungal Genome Extraction Kit purchased from Hangzhou Biospin Technology Co., Ltd.). The specific operations are as follows:

[0074] a: The mortar, pestle and medicine spoon were sterilized in advance, and the test bench was wiped with 75% alcohol. Pour some liquid nitrogen into a mortar and let the mortar and pestle pre-cool. Then, take out the Aspergillus flavus mycelium suspended in the liquid medium and pour it into a mortar, smash and grind the fung...

Embodiment 3F

[0113] Example 3 FITC@AuNCs-LAMP sensitivity detection and establishment of standard curve

[0114] (1) Extract the genome

[0115] According to the method described in step (1) of Example 2, the genomes of Aspergillus niger, Aspergillus flavus, Aspergillus aegypti, Penicillium viridis and Fusarium oxysporum were extracted respectively, the concentration of the obtained Aspergillus niger genome was 7.2ng / μL, the concentration of Aspergillus flavus It was 7.2ng / μL, the genome concentration of Aspergillus aegypti was 6.9ng / μL, the genome concentration of Penicillium aeruginosa was 3.3ng / μL, and the genome concentration of Fusarium oxysporum was 14ng / μL.

[0116] (2) Optimization of FITC@AuNCs-LAMP reaction conditions

[0117] The amplification conditions of the FITC@AuNCs-LAMP reaction were explored, and the reaction temperature was 55°C-65°C and the reaction time was 45min-70min for electrophoresis verification. The result is as Figure 5 As shown, there are bands running ou...

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PUM

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Abstract

The invention relates to a ratio fluorescence biosensor based on gold nanoclusters, a preparation method of the ratio fluorescence biosensor and application of the ratio fluorescence biosensor to detection of aspergillus mycetes. The method comprises the following steps: by taking an aspergillus mould DNA genome as a target, designing an LAMP primer, and adding FITC-coated AuNCs on the basis of determining the specificity of the primer to carry out LAMP reaction. AuNCs protected by BSA is used as a fluorophore, FITC is used as a pH response signal, along with LAMP reaction, a large number of pyrophosphate and hydrogen ions are released, the ratio fluorescence signals of FITC and AuNCs are gradually reduced, and the method can be used for quantitative analysis of aspergillus mould detection.

Description

technical field [0001] The invention relates to the field of food safety biotechnology, in particular to a ratio fluorescent biosensor based on gold nanoclusters, a preparation method thereof, and its application in detecting Aspergillus. Background technique [0002] Leisure food is the food that people eat during breaks and breaks. It is mainly divided into cereal products, dried fruit products, confectionary products and meat products. In 2020, the total output value of the snack food industry will reach 3 trillion yuan, and it is expected to exceed 4 trillion yuan in 2025. It can be seen that the snack food industry is developing rapidly and has huge development potential. Among them, the annual output value of the roasted nuts industry has rapidly increased from 45.2 billion yuan to 121.4 billion yuan, with an average annual compound growth rate of 15.2%, which is much higher than the overall snack food. At the same time, since 2017, all parts of the country have succe...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11C12R1/67C12R1/685C12R1/66C12R1/77C12R1/80
CPCC12Q1/6895C12Q1/6844C12Q2531/119C12Q2545/113C12Q2563/107C12Q2565/607
Inventor 王周平潘柯文顾千辉杨宁茹尚滋萱贺逸菲马鹏飞
Owner JIANGNAN UNIV
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