Fluorescence resonance energy transfer sensor based on MoWS2 nanoparticles, construction and rapid aspergillus flavus detection method

Abstract

The invention belongs to the field of biological detection, and particularly relates to a fluorescence resonance energy transfer sensor based on MoWS2 nanoparticles, construction of the fluorescence resonance energy transfer sensor and a rapid aspergillus flavus detection method. The fluorescence resonance energy transfer sensor based on the MoWS2 nanoparticles, provided by the invention, comprises PAA-MoWS2 nanoparticles and EuNPs-probe DNA (Deoxyribose Nucleic Acid) formed by modifying EuNPs with a DNA (Deoxyribose Nucleic Acid) probe, wherein the DNA probe is a probe for specifically recognizing aspergillus flavor-1. The rapid and high-sensitivity detection on the aspergillus flavus can be realized; the method is simple to operate, rapid in detection and suitable for rapid detection and analysis of aspergillus flavus.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and specifically relates to a fluorescence resonance energy transfer sensor based on MoWS2 nanoparticles, and a construction and a rapid detection method for Aspergillus flavus. Background Art

[0002] Aflatoxin is the most toxic and carcinogenic natural pollutant discovered so far. For example, aflatoxin B1 is 10 times more toxic than potassium cyanide and 68 times more toxic than arsenic. In 1993, it was classified as a Class I carcinogen by the Cancer Research Institute of the World Health Organization (WHO). According to statistics, the global food loss due to aflatoxin and other fungal toxins is as high as 25% each year. Over the years, poisoning and death incidents caused by aflatoxin in humans and animals have occurred frequently in various countries around the world, which not only seriously threatens the health and life safety of humans and animals, but also easily causes social panic. Therefore, it has attracted great attention from countries around the world. Aflatoxin is mainly produced by Aspergillus fungi such as Aspergillus flavus and Aspergillus parasiticus. If aflatoxin-producing fungi can be detected and identified in time, and necessary measures can be taken to intervene in toxin pollution in agricultural products as soon as possible, it will be of great significance to reduce economic losses and improve the quality and safety of grain and oil products.

[0003] At present, the commonly used detection methods for aflatoxin-producing fungi at home and abroad are traditional morphological identification, culture medium identification, PCR detection, etc. These methods are cumbersome and have long detection cycles, which are difficult to meet the daily real-time rapid monitoring needs. DNA sensors have the advantages of simple operation, high sensitivity, strong specificity, fast detection speed and easy miniaturization, and have become a research hotspot for the detection of microbial pathogens. The nor-1 gene involved in the aflatoxin biosynthesis pathway is also called the aflD gene. It encodes a catalytic enzyme with a molecular weight of 29kDa and plays an important role in the synthesis of aflatoxin. At present, there are literature reports on the detection of nor-1 gene electrochemical sensors, but the electrode modification process is cumbersome, and the repeatability and stability between electrodes need to be improved. Therefore, the construction of a DNA homogeneous sensor based on fluorescence resonance energy transfer to achieve simple, rapid and highly sensitive detection of Aspergillus flavus can not only provide new ideas for the detection of aflatoxin-producing fungi, but also provide key technical support for the detection and early warning control research of microbial pathogens.

[0004] Rare earth europium fluorescent nanomaterials have the advantages of long fluorescence lifetime, large Stokes shift, and high fluorescence quantum yield, and have been used as signal markers for fluorescent biological probes. When constructing fluorescence energy transfer sensors, the fluorescence quenching properties of energy acceptors have an important influence on the detection sensitivity.

[0005] In recent years, transition metal dichalcogenide alloy MoWS2 has attracted extensive attention in the field of optoelectronics due to its unique optical and electronic properties. Recently, MoWS2 has been used to construct electrochemical sensors for analytical detection applications, but MoWS2 nanosheets are dispersed in ethanol solutions. It still does not have good dispersibility in aqueous solutions, which greatly limits the application of MoWS2 nanomaterials in the field of biosensing.

[0006] The present invention proposes a one-step method for preparing water-soluble MoWS2 nanoparticles, and uses them as energy acceptors to quench the fluorescence of rare earth europium fluorescent nanoparticles. A new type of fluorescence resonance energy transfer sensor is constructed based on the van der Waals force between single-stranded DNA and MoWS2 nanoparticles, and highly sensitive and rapid detection of Aspergillus flavus is achieved. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a fluorescence resonance energy transfer sensor based on MoWS2 nanoparticles, its construction and application. It can realize rapid and highly sensitive detection of Aspergillus flavus; it is simple to operate and has rapid detection, and is suitable for rapid detection and analysis of Aspergillus flavus.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a fluorescence resonance energy transfer sensor based on MoWS2 nanoparticles, comprising PAA-MoWS2 nanoparticles and EuNPs-probe DNA formed by modifying EuNPs with DNA probes, wherein the DNA probe is a probe that specifically recognizes Aspergillus flavus nor-1.

[0010] According to the above scheme, the DNA probe sequence used was: 5'-CAACCCGCCTGATGCTGCAGCAGTC-3'.

[0011] According to the above scheme, PAA-MoWS2 nanoparticles are prepared into an aqueous solution for use; DNA probe-modified EuNPs are prepared into a solution for use.

[0012] According to the above scheme, the amount of PAA-MoWS2 nanoparticles is used to satisfy the requirement that the fluorescence emission intensity of EuNPs-probe DNA is quenched by more than 70%, preferably more than 80%. The degree of quenching affects the response degree, signal-to-noise ratio, etc. A certain degree of quenching has a better signal-to-noise ratio and responsiveness, and can be used for aflatoxin fluorescence detection to more sensitively and accurately detect the content of aflatoxin in the sample.

[0013] According to the above scheme, the DNA probe is modified with amino groups, the surface of EuNPs is modified with carboxyl groups, and the amino-modified DNA probe is coupled with EuNPs with carboxyl groups on the surface to obtain EuNPs-probe DNA.

[0014] The second aspect of the present invention provides a method for preparing the above-mentioned fluorescence resonance energy transfer sensor based on MoWS2 nanoparticles, comprising the following steps:

[0015] 1) Provide EuNPs for backup;

[0016] 2) Providing PAA-MoWS2 nanoparticles for backup;

[0017] 3) Modification of probe DNA: providing EuNPs with surface carboxyl activated, performing probe DNA modification, and obtaining modified EuNPs-probe DNA, wherein the DNA probe is a probe that specifically recognizes Aspergillus flavus nor-1.

[0018] According to the above scheme, the preparation method of the PAA-MoWS2 nanoparticles is:

[0019] Preparation of MoWS2 nanoparticles: under constant stirring, add CH4N2S and C2H2O4·2H2O to the solution of sodium molybdate and sodium tungstate, heat at 200-220℃ for 10-14h, and post-treat to obtain MoWS2;

[0020] MoWS2 was added to the PAA solution (PAA:MoWS2=1:1-3, mass ratio), and ultrasonicated at room temperature to obtain a black dispersion. The dispersion was centrifuged at 3000-5000 rpm to remove large particles. The supernatant was centrifuged to obtain a precipitate, water was added, and the mixture was washed by centrifugation at 10000-12000 rpm. The precipitate was collected as PAA-MoWS2, vacuum dried, dispersed in water, and stored at room temperature for later use.

[0021] According to the above scheme, Na2MoO4 is in excess relative to Na2WO4. Preferably, the molar ratio of Na2MoO4:Na2WO4 is 26:1-20:1, and the molar ratio of Na2WO4:CH4N2S:C2H2O4 is 1:60-100:10-20.

[0022] According to the above scheme, the modification steps of the probe DNA are: adding probe DNA to the EuNPs activated by surface carboxyl groups, reacting overnight, and post-processing to obtain EuNPs-probe DNA. The post-processing is: adding tris (hydroxymethylaminomethane) (Tris) to terminate the reaction, centrifuging and washing with water.

[0023] According to the above scheme, the preparation method of EuNPs with surface carboxyl activated is as follows: the EuNPs prepared in step 1 are dispersed in morpholineethanesulfonic acid (MES) buffer, 1-ethyl-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) are added, and the precipitate is collected by centrifugation after incubation at room temperature.

[0024] According to the above scheme, the ratio of the mass of EuNPs and the amount of probe DNA added was 2-6 mg:2-6 nmol, and the concentrations of EDC and NHS were 0.4-1 mM and 1-3 mM, respectively.

[0025] According to the above scheme, the prepared EuNPs-probe DNA was dispersed in HEPES buffer for later use.

[0026] The third aspect of the present invention provides a rapid detection method for Aspergillus flavus based on the above-mentioned fluorescence resonance energy transfer sensor, extracting the Aspergillus flavus DNA of the sample to be tested, PCR amplifying nor-1, incubating with a solution of EuNPs-probe DNA, adding an aqueous solution of MoWS2 nanoparticles, and measuring the fluorescence emission intensity under 360nm excitation. Based on the standard curve of the fluorescence recovery degree, i.e. (F-F0) / F0 and the concentration of Aspergillus flavus spores, F0: the initial fluorescence emission intensity in the absence of sample, F: the fluorescence emission intensity measured when the sample is present, the Aspergillus flavus spore concentration of the sample to be tested is analyzed and obtained.

[0027] According to the above scheme, the incubation time is 20 min to 40 min.

[0028] According to the above scheme, EuNPs-probe DNA is dispersed in HEPES buffer for use at a concentration of 0.01-0.03 mg / mL, preferably 0.015-0.025 mg / mL; PAA-MoWS2 nanoparticles are prepared into aqueous solution for use at a concentration of 0.005 mg / mL-0.04 mg / mL, preferably 0.025 mg / mL-0.04 mg / mL. The amount of EuNPs-probe DNA used affects the initial fluorescence emission intensity value of F0, and the amount of MoWS2 used affects the degree of quenching, which may affect the degree of response, signal-to-noise ratio, etc. A certain dosage concentration has a better signal-to-noise ratio and responsiveness, and has a more excellent sensitivity for aflatoxin detection.

[0029] According to the above scheme, the method for constructing the standard curve of fluorescence emission intensity and Aspergillus flavus spore concentration is as follows: using Aspergillus flavus toxin-producing standard bacteria to obtain Aspergillus flavus spore solutions of different concentrations, inoculating spore solutions of different concentrations into peanuts, and making the final spore concentration be 10 2 -10 7 / g, after incubation, the total DNA was extracted, amplified by PCR, and incubated with the EuNPs-probeDNA solution, and then an aqueous solution of MoWS2 nanoparticles was added to measure the fluorescence emission intensity under 360nm excitation, and the standard curve of the fluorescence recovery degree, i.e. (F-F0) / F0 and the Aspergillus flavus spore concentration, was established through the correlation between the Aspergillus flavus spore concentration and the fluorescence recovery degree.

[0030] The specific method is as follows: first, the standard strain of Aspergillus flavus 3.4408 was inoculated on Czapek-Dox Agar (CDA) solid medium for culture, and after culturing at 28°C and 90% humidity for 7 days, the spores were washed with 5% glycerol / water, and then the spores were counted by the hemocytometer. A laboratory grinder was used to grind 10g of blank peanut sample into peanut powder with a diameter of less than 500μm. Subsequently, 0.2g was accurately weighed and transferred to a 2.0mL test tube without nuclease. The spore solution obtained above was diluted 10 times to 10 2 -10 7 200 μL of spore solution of different concentrations was inoculated into peanuts to make the final spore concentration be 10 2 -10 7 After incubation at 28°C for 1 hour, total DNA was extracted by CTAB method, amplified by PCR and detected by fluorescence sensor. The fluorescence emission intensity at 615nm was measured by fluorescence spectrophotometer under 360nm excitation, and the standard curve for detecting Aspergillus flavus in peanuts was established by the correlation between the concentration of Aspergillus flavus spores and the degree of fluorescence recovery, i.e. (F-F0) / F0.

[0031] The present invention selects rare earth europium fluorescent nanoparticles EuNPs as energy donors, PAA modified MoWS2 nanoparticles as energy acceptors, probe DNA is coupled with EuNPs to obtain EuNPs-probe DNA, and the effect of DNA probe and MoWS2 nanoparticles closes the distance between the two to promote the generation of fluorescence resonance energy transfer, resulting in fluorescence resonance energy transfer. When the target DNA or the Aspergillus flavus nor-1 gene (a sample containing Aspergillus flavus) appears, it is hybridized with the probe DNA to form double-stranded DNA, which weakens the effect of the probe DNA and the MoWS2 nanoparticles, so that the distance between EuNPs and the MoWS2 nanoparticles becomes larger, resulting in the inhibition of fluorescence resonance energy transfer, thereby restoring the fluorescence of EuNPs, thereby establishing a fluorescence sensor based on the fluorescence recovery mechanism. It has the characteristics of strong binding force with the target molecule, high sensitivity, good selectivity, and is suitable for the detection of Aspergillus flavus in actual samples. Through the change of fluorescence emission intensity (correlation of fluorescence recovery degree with target DNA or Aspergillus flavus spore concentration), rapid and highly sensitive detection of Aspergillus flavus is achieved.

[0032] The beneficial effects of the present invention are:

[0033] The fluorescence resonance energy transfer sensor of the present invention is a fluorescence sensor based on the fluorescence mechanism of fluorescence resonance energy transfer and target specific recognition. It selects rare earth europium fluorescent nanoparticles as energy donors and PAA-modified MoWS2 nanoparticles as energy acceptors. The distance between DNA probes and MoWS2 nanoparticles is shortened through the action of the two to promote the generation of fluorescence resonance energy transfer. The target DNA or the nor-1 gene of Aspergillus flavus is hybridized to the probe DNA through specific complementary pairing to form a DNA double helix structure, weakening the action between the single-stranded DNA probe and the MoWS2 nanoparticles, thereby increasing the distance between the energy donor-acceptor pair, hindering the fluorescence resonance energy transfer process and causing the fluorescence recovery of EuNPs. The detection of Aspergillus flavus is achieved through the change of the fluorescence recovery intensity. The method is simple to operate and rapid in detection, and is suitable for rapid detection and analysis.

[0034] The present invention prepares MoWS2 nanoparticles with good water solubility in a one-step method, and for the first time uses MoWS2 nanoparticles with good fluorescence quenching ability and biocompatibility as energy donors; and uses EuNPs with high fluorescence quantum yield and good fluorescence performance as energy donors, and constructs a fluorescence resonance energy transfer sensor based on the principle of fluorescence resonance energy transfer and the principle of DNA complementary pairing. The aflatoxin detection method provided by the present invention detects aflatoxin, which is expected to provide key technical support for inhibiting aflatoxin and its toxin-producing fungi from the source, and has broad application prospects in aflatoxin early warning control research and other food contaminant detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 (A) Transmission electron microscopy characterization of PAA-MoWS2 nanoparticles; (B) PAA-MoWS2 aqueous dispersion.

[0036] Figure 2 Schematic diagram of the overlap between the fluorescence emission spectrum of EuNPs and the UV spectrum of MoWS2 nanoparticles.

[0037] Figure 3 (A) Fluorescence spectra of 0.02 mg / mL EuNPs-probe DNA after incubation with different concentrations of PAA-MoWS2; (B) Fluorescence spectra of 0.02 mg / mL EuNPs before and after incubation with 0.04 mg / mL PAA-MoWS2.

[0038] Figure 4 Standard curve of fluorescence emission intensity of target DNA at different concentrations. F0, fluorescence emission intensity when there is no target DNA, F, fluorescence emission intensity when there are different concentrations of target DNA.

[0039] Figure 5Standard curve of fluorescence emission intensity of Aspergillus flavus spores at different concentrations. F0, fluorescence emission intensity when the concentration of Aspergillus flavus spores is 0, F, fluorescence emission intensity at different concentrations of Aspergillus flavus spores. DETAILED DESCRIPTION

[0040] In order to enhance the understanding of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and examples. The following examples are mainly for further illustrating the contents of the present invention, but should not be construed as limitations of the present invention. Without departing from the essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0041] Example 1

[0042] Construction of fluorescence resonance energy transfer sensor:

[0043] 1) Preparation of rare earth europium fluorescent nanoparticles for use:

[0044] The specific preparation process is as follows: styrene monomer (10 mM) and acrylic acid monomer (0.95 mM) are dissolved in 10 mL of deionized water containing 0.45 mM sodium dodecyl sulfate (SDS), added to a round-bottom flask, and stirred evenly with a magnetic stirrer. Then, the air in the round-bottom flask is removed with high-purity nitrogen and heated to 70°C. Subsequently, 0.5 mL of 0.15 mM potassium persulfate is added to the flask, stirred for 8 hours, cooled to room temperature, and filtered with a whatman 2v filter paper (pore size 8 μm) to collect the polystyrene nanoparticles with carboxyl groups, and dialyzed and purified with a dialysis bag (molecular weight cutoff 30,000 Da). The polystyrene nanoparticles prepared above were diluted with 10 mL of acetone water (v / v=1:1), and then 100 μL of 0.1 M europium trichloride, 300 μL of trioctylphosphine oxide (TOPO), 100 μL of phenanthroline and 400 μL of 0.1 M β-diketone (β-NTA) were added, and the mixture was stirred and reacted at 60°C in the dark for 10 h, then cooled to room temperature and continued to react for 2 h. Finally, the organic solvent in the solution was removed by vacuum distillation, and the solution was dialyzed with deionized water for 5 days to remove the remaining small molecules. The liquid in the dialysis bag was collected and stored at 4°C after adding 0.05% sodium azide.

[0045] 2) Preparation of EuNPs-probe DNA;

[0046] 2 mg of EuNPs prepared in step 1 were ultrasonically dispersed in 2 mL of morpholineethanesulfonic acid (MES, pH = 5.5, 10 mM) buffer, 0.4 mM 1-ethyl-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC·HCl) and 1 mM N-hydroxysuccinimide (NHS) were added, and the precipitate was collected by centrifugation after incubation at room temperature for 40 min to obtain EuNPs with surface carboxyl activated, 2 nmol of probe DNA was added, and the reaction was allowed to proceed overnight at room temperature, and 10 mg of tris(hydroxymethyl)aminomethane (Tris) was added to terminate the reaction to obtain a DNA probe modified with EuNPs, which was recorded as EuNPs-probe DNA, and then centrifuged and washed with water, and finally the EuNPs-probe was dispersed in 2 mL of HEPES (pH = 7.4, 10 mM) buffer for use;

[0047] Probe used: 5'-NH2-CAACCCGCCTGATGCTGCAGCAGTC-3'

[0048] 2) Preparation of PAA-modified MoWS2 nanoparticles for later use:

[0049] The specific preparation process is as follows: First, prepare MoWS2 nanoparticles. Dissolve 3.85mmol Na2MoO4·2H2O and 0.15mmol Na2WO4·2H2O in 40mL deionized water. Then, add 15mmol CH4N2S and 3mmol C2H2O4·2H2O under constant stirring. After the precursor solution is converted into a transparent liquid, it is transferred to a 60mL high-pressure reactor and heated at 200℃ for 12h. After cooling to room temperature, wash it three times with high-purity water and ethanol respectively, and then dry it in a vacuum drying oven. Then prepare polyacrylic acid (PAA)-modified MoWS2 nanoparticles (PAA-MoWS2). Weigh 20 mg of PAA and place it in a 100 mL round-bottom flask filled with 20 mL of ultrapure water. Stir evenly, then add 20 mg of MoWS2 powder and ultrasonicate at room temperature for 12 h to obtain a black dispersion. Centrifuge at 3000 rpm for 10 min to remove large particles. Wash the obtained supernatant with water three times by centrifugation (10000 rpm, 10 min), dry in a vacuum at 60 °C, disperse in high-purity water at a concentration of 1 mg / mL, and store at room temperature for later use.

[0050] Transmission electron microscopy characterization of PAA-MoWS2 nanoparticles Figure 1 (A) shows; PAA-MoWS2 aqueous dispersion is as shown Figure 1 (B) is shown. Schematic diagram of the overlap between the fluorescence emission spectrum of EuNPs and the ultraviolet spectrum of MoWS2 nanoparticles is shown in Figure 2 shown.

[0051] 3) Based on the construction method of MoWS2 fluorescence resonance energy transfer sensor, 0.02 mg / mL EuNPs-probeDNA was incubated with different concentrations of PAA-MoWS2 nanoparticles in HEPES buffer (10 mM, pH 7.4) and the fluorescence emission intensity (peak fluorescence intensity) at an excitation wavelength of 360 nm was measured. As the concentration of PAA-MoWS2 increased, the fluorescence quenching efficiency gradually increased. When the concentration of PAA-MoWS2 was 0.02-0.04 mg / mL, the fluorescence quenching efficiency was more than 70%, and when the concentration of PAA-MoWS2 was 0.04 mg / mL, the fluorescence quenching efficiency reached 87%. Figure 3 (A) shows that within this concentration range, the nonspecific quenching caused by the incubation of PAA-MoWS2 with EuNPs is almost negligible, where: The fluorescence spectra before and after incubation of 0.02 mg / mL EuNPs with 0.04 mg / mL PAA-MoWS2 are shown in Figure 3 (B) As shown below, the concentration of MoWS2 nanoparticles was selected as 0.04 mg / mL for the fluorescence recovery experiment.

[0052] Example 2 Target DNA Detection:

[0053] After incubating the target DNA standard solutions with different concentrations (concentrations were 0, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 3 nM, and 5 nM, respectively) with 0.02 mg / mL EuNPs-probe DNA, 0.04 mg / mL MoWS2 nanoparticles were added, and the fluorescence emission intensity of the solution was measured at 615 nm using a fluorescence spectrophotometer, and a standard curve of F-F0 / F0 and target DNA concentration was established. When there was no target DNA, the fluorescence emission intensity measured after incubation of 0.02 mg / mL EuNPs-probe DNA with 0.04 mg / mL PAA-MoWS2 was F0, as shown in FIG. Figure 4 shown.

[0054] Target DNA used: 5'-GACTGCTGCAGCATCAGGCGGGTTG-3'

[0055] Example 3 Peanut aflatoxin sample detection:

[0056] The constructed fluorescent sensor was used to detect Aspergillus flavus samples in peanuts. The detection process is as follows: the standard strain of Aspergillus flavus 3.4408 was inoculated on Czapek-Dox Agar (CDA) solid culture medium and cultured at 28°C and 90% humidity for 7 days. The spores were rinsed with 5% glycerol / water and then counted by hemocytometer. Use a laboratory grinder to grind 10g of blank peanut sample into peanut powder with a diameter of less than 500μm. Subsequently, 0.2g was accurately weighed and transferred to a 2.0mL test tube without nuclease. The spore solution obtained above was diluted 10 times to 10 2 -10 7 200 μL of spore solution of different concentrations was inoculated into peanuts to make the final spore concentration be 10 2 -10 7 After incubation at 28°C for 1 hour, the total DNA was extracted by CTAB method, amplified by PCR, and the fluorescence emission intensity under 360nm excitation was measured by a fluorescence spectrophotometer using the same method as in Example 2. The standard curve for detecting aflatoxin in peanuts was established by the correlation between the concentration of aflatoxin spores and the degree of fluorescence recovery. The detection standard curve established with (F-F0) / F0 as the ordinate and the concentration of aflatoxin spores as the abscissa is shown in the figure. Figure 5 shown.

[0057] F0, the fluorescence emission intensity when the Aspergillus flavus spore concentration is 0, that is, the fluorescence emission intensity measured after incubation of 0.02 mg / mL EuNPs-probe DNA with 0.04 mg / mL PAA-MoWS2.

[0058] The standard curve equation for detecting Aspergillus flavus in peanuts is: y=-0.04759+0.10819x, and there is a good linear relationship between the fluorescence recovery degree (F-F0) / F0 and the number of Aspergillus flavus spores.

[0059] Primer used for PCR amplification: nor1-F: 5'-TGCTCCCGTCCTACTGTTTC-3'

[0060] nor1-R:5'-CGCCCATATCAGTTTTGCAC-3'

[0061] Sample addition recovery rate determination. 10g of blank peanut sample was inoculated with spores of Aspergillus flavus standard strain 3.4408 to a concentration of 10 3 pcs / g, 10 4 pcs / g, 10 6 / g. After inoculation, incubate at 37°C for 1h, followed by total DNA extraction and PCR amplification, and then use the above-mentioned biosensor to detect the PCR amplification product to obtain the aflatoxin content in the sample. The results of the added recovery rate determination are shown in Table 1. Comprehensive analysis of the intra-group and inter-group determination results shows that the added recovery rate of aflatoxin spores is 95-109%, and the coefficient of variation is 0.35-7.3%. The added recovery rate is high and the coefficient of variation is small, indicating that this method has good reproducibility and accuracy in detecting aflatoxin. This method detects aflatoxin-producing genes from the source, which is suitable for rapid detection and early warning analysis.

[0062] Table 1 Results of the fluorescence sensor detection of peanut aflatoxin spike experiment

[0063]

Claims

1. A fluorescence resonance energy transfer sensor based on MoWS2 nanoparticles, characterized in that: The invention comprises EuNPs-probe DNA formed by modifying EuNPs with PAA-MoWS2 nanoparticles and DNA probes, wherein the DNA probe is a probe which specifically identifies Aspergillus flavus nor-1.

2. The fluorescence resonance energy transfer sensor according to claim 1, characterized in that: The DNA probe sequence used was: 5'-CAACCCGCCTGATGCTGCAGCAGTC-3'.

3. The fluorescence resonance energy transfer sensor according to claim 1, characterized in that: PAA-MoWS2 nanoparticles are prepared into an aqueous solution for use; DNA probes are modified with EuNPs and prepared into a solution for use; the DNA probes are modified with amino groups, the surface of EuNPs is modified with carboxyl groups, and the amino-modified DNA probes are coupled with EuNPs with carboxyl groups on the surface to obtain EuNPs-probe DNA.

4. The fluorescence resonance energy transfer sensor according to claim 1, characterized in that: The amount of PAA-MoWS2 nanoparticles used was such that the fluorescence emission intensity of EuNPs-probe DNA was quenched by more than 70%.

5. The method for preparing the fluorescence resonance energy transfer sensor based on MoWS2 nanoparticles according to claim 1, characterized in that: The following steps are involved: 1) Provide EuNPs for backup; 2) Providing PAA-MoWS2 nanoparticles for backup; 3) Modification of probe DNA: providing EuNPs with surface carboxyl activated, performing probe DNA modification, and obtaining modified EuNPs-probe DNA, wherein the DNA probe is a probe that specifically recognizes Aspergillus flavus nor-1.

6. The preparation method according to claim 5, characterized in that: The following steps are involved: The preparation method of the PAA-MoWS2 nanoparticles is as follows: Preparation of MoWS2 nanoparticles: under constant stirring, add CH4N2S and C2H2O4·2H2O to the solution of sodium molybdate and sodium tungstate, heat at 200-220℃ for 10-14h, and post-treat to obtain MoWS2; MoWS2 was added to the PAA solution at a mass ratio of PAA:MoWS2 = 1:1-3, and ultrasonicated at room temperature to obtain a black dispersion. The dispersion was centrifuged at 3000-5000 rpm to remove large particles. The supernatant was centrifuged to obtain a precipitate, water was added, and the solution was washed by centrifugation at 10000-12000 rpm. The precipitate was collected as PAA-MoWS2, vacuum dried, dispersed in water, and stored at room temperature for later use.

7. The preparation method according to claim 5, comprising the following steps: The modification steps of the probe DNA are as follows: adding the probe DNA to the EuNPs activated with surface carboxyl groups, reacting overnight, and post-processing to obtain the EuNPs-probe DNA.

8. A method for rapid detection of Aspergillus flavus for non-disease diagnosis and treatment purposes based on the fluorescence resonance energy transfer sensor of claim 1, characterized in that: The Aspergillus flavus DNA of the sample to be tested is extracted, and after PCR amplification of nor-1, it is incubated with the solution of EuNPs-probe DNA in the fluorescence resonance energy transfer sensor according to claim 1, and then an aqueous solution of MoWS2 nanoparticles is added to obtain the fluorescence emission intensity under 360nm excitation. Based on the standard curve of the fluorescence recovery degree, i.e. (F-F0) / F0 and the concentration of Aspergillus flavus spores, F0: the initial fluorescence emission intensity when there is no sample to be tested, F: the fluorescence emission intensity measured when the sample to be tested is present, the Aspergillus flavus spore concentration of the sample to be tested is analyzed and obtained.

9. The method for rapid detection of Aspergillus flavus according to claim 8, characterized in that: EuNPs-probe DNA was dispersed in HEPES buffer and used at a concentration of 0.015-0.025 mg / mL; PAA-MoWS2 nanoparticles were prepared into aqueous solution and used at a concentration of 0.025 mg / mL-0.04 mg / mL.

10. The method for rapid detection of Aspergillus flavus according to claim 9, characterized in that: The method for constructing the standard curve of fluorescence emission intensity and Aspergillus flavus spore concentration is as follows: using Aspergillus flavus toxin-producing standard bacteria to obtain Aspergillus flavus spore solutions of different concentrations, inoculating the spore solutions of different concentrations into peanuts, and making the final spore concentration be 10 2 -10 7 / g, after incubation, the total DNA was extracted, amplified by PCR, and incubated with the EuNPs-probe DNA solution, and then an aqueous solution of MoWS2 nanoparticles was added to measure the fluorescence emission intensity under 360nm excitation, and the standard curve of the fluorescence recovery degree, i.e. (F-F0) / F0 and the Aspergillus flavus spore concentration, was established through the correlation between the Aspergillus flavus spore concentration and the fluorescence recovery degree.

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