A fluorescence resonance energy transfer sensor based on MoWS2 nanoparticles, its construction, and a rapid detection method for Aspergillus flavus.

Abstract

The application belongs to the field of biological detection, and particularly relates to a fluorescence resonance energy transfer sensor based on MoWS2 nanoparticles, construction and a rapid detection method of aspergillus flavus. The fluorescence resonance energy transfer sensor based on MoWS2 nanoparticles provided by the application comprises PAA-MoWS2 nanoparticles and EuNPs-probe DNA formed by DNA probe modified EuNPs, wherein the DNA probe is a probe capable of specifically recognizing nor-1 of aspergillus flavus. The sensor can realize rapid and high-sensitivity detection of aspergillus flavus, and is simple to operate, rapid in detection and suitable for rapid detection and analysis of aspergillus flavus.

Description

Technical Field

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

[0002] Aflatoxin is the most toxic and carcinogenic natural contaminant discovered to date. 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 Group 1 carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO). Statistics show that global food losses due to aflatoxin and other fungal toxins reach as high as 25% annually. For many years, serious incidents of poisoning and death in humans and animals caused by aflatoxin have occurred frequently worldwide, not only severely threatening human and animal health and safety but also easily triggering social panic, thus attracting great attention from countries around the world. Aflatoxin is mainly produced by Aspergillus fungi such as Aspergillus flavus and Aspergillus parasiticus. Timely detection and identification of aflatoxin-producing fungal contamination, and early intervention with necessary measures to prevent toxin contamination in agricultural products, are of great significance for reducing economic losses and improving the quality and safety of grain and oil products.

[0003] Currently, common detection methods for aflatoxin-producing fungi, both domestically and internationally, include traditional morphological identification, culture medium identification, and PCR detection. These methods are cumbersome and time-consuming, making them unsuitable for routine, real-time, and rapid monitoring. DNA sensors, with their advantages of simple operation, high sensitivity, strong specificity, fast detection speed, and ease of miniaturization, have become a research hotspot in the detection of pathogenic microorganisms. The nor-1 gene, also known as the aflD gene, is involved in the aflatoxin biosynthesis pathway and encodes a 29 kDa catalytic enzyme that plays a crucial role in aflatoxin synthesis. While electrochemical sensors based on the nor-1 gene have been reported in the literature, the electrode modification process is cumbersome, and the repeatability and stability between electrodes need improvement. Therefore, constructing a homogeneous DNA sensor based on fluorescence resonance energy transfer (FRET) to achieve simple, rapid, and highly sensitive detection of Aspergillus flavus not only provides a new approach for the detection of aflatoxin-producing fungi but also offers key technical support for the detection and early warning control research of pathogenic microorganisms.

[0004] Rare-earth europium fluorescent nanomaterials possess advantages such as long fluorescence lifetime, large Stokes shift, and high fluorescence quantum yield, and have been used as signal biomarkers for fluorescent biological probes. In the construction of fluorescent energy transfer sensors, the fluorescence quenching properties of the energy acceptor have a significant impact on detection sensitivity.

[0005] In recent years, MoWS2, a transition metal dichalcogenide alloy, has attracted widespread attention in the optoelectronic field due to its unique optical and electronic properties. Recently, MoWS2 has been used to construct electrochemical sensors for analytical detection applications; however, MoWS2 nanosheets are dispersed in ethanol solutions. They still do not exhibit good dispersibility in aqueous solutions, which greatly limits the application of MoWS2 nanomaterials in biosensing.

[0006] This invention proposes a one-step method for preparing water-soluble MoWS2 nanoparticles, which are then used as energy acceptors to quench the fluorescence of rare-earth europium fluorescent nanoparticles. A novel fluorescent resonance energy transfer sensor is constructed based on the van der Waals forces between single-stranded DNA and MoWS2 nanoparticles, enabling highly sensitive and rapid detection of Aspergillus flavus. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a fluorescence resonance energy transfer sensor based on MoWS2 nanoparticles, its construction, and its application. It enables rapid and highly sensitive detection of Aspergillus flavus; it is simple to operate, provides rapid detection, and is suitable for the rapid detection and analysis of Aspergillus flavus.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[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 modified with a DNA probe, 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 is: 5'-CAACCCGCCTGATGCTGCAGCAGTC-3'.

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

[0012] According to the above scheme, the amount of PAA-MoWS2 nanoparticles used is such 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 level, signal-to-noise ratio, etc. A certain degree of quenching results in a better signal-to-noise ratio and responsiveness, which can more sensitively and accurately detect the content of Aspergillus flavus in the sample.

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

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

[0015] 1) Provide EuNPs as backup;

[0016] 2) Provide PAA-MoWS2 nanoparticles for later use;

[0017] 3) Modification of probe DNA: EuNPs with surface carboxyl activated are provided for probe DNA modification to obtain modified EuNPs-probe DNA. 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 as follows:

[0019] Preparation of MoWS2 nanoparticles: CH4N2S and C2H2O4·2H2O were added to a solution of sodium molybdate and sodium tungstate under constant stirring, and the mixture was heated at 200-220℃ for 10-14 h. MoWS2 was obtained after post-treatment.

[0020] Add MoWS2 to the PAA solution (PAA:MoWS2 = 1:1-3, mass ratio), sonicate at room temperature to obtain a black dispersion, centrifuge at 3000-5000 rpm to remove large particles, centrifuge the supernatant to obtain a precipitate, add water, wash by centrifugation at 10000-12000 rpm, collect the precipitate to obtain PAA-MoWS2, vacuum dry, disperse in water, and store 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 to Na2WO4 is 26:1 to 20:1, and the molar ratio of Na2WO4 to CH4N2S to C2H2O4 is 1:60-100:10-20.

[0022] According to the above scheme, the modification steps of the probe DNA are as follows: probe DNA is added to EuNPs with surface carboxyl-activated groups, reacted overnight, and post-processed to obtain EuNPs-probe DNA. The post-processing is as follows: the reaction is terminated by adding tris(hydroxymethyl)aminomethane (Tris), followed by centrifugation and washing with water.

[0023] According to the above scheme, the preparation method of surface carboxyl-activated EuNPs is as follows: the EuNPs prepared in step 1 are dispersed in morpholine ethanesulfonic acid (MES) buffer, 1-ethyl-(3-dimethylaminopropyl)-carbonyldiimide 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 EuNPs mass to probe DNA added is 2-6 mg: 2-6 nmol, and the concentrations of EDC and NHS are 0.4-1 mM and 1-3 mM, respectively.

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

[0026] The third aspect of this invention provides a rapid detection method for Aspergillus flavus based on the above-mentioned fluorescence resonance energy transfer sensor. The method involves extracting Aspergillus flavus DNA from the sample to be tested, amplifying it with PCR (nor-1), incubating it with a solution of EuNPs-probe DNA, adding an aqueous solution of MoWS2 nanoparticles, and measuring the fluorescence emission intensity under 360 nm excitation. Based on a standard curve of fluorescence recovery (F-F0) / F0 versus Aspergillus flavus spore concentration, where F0 is the initial fluorescence emission intensity without a sample and F is the fluorescence emission intensity measured in the presence of the sample, the Aspergillus flavus spore concentration of the sample to be tested is 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 at a concentration of 0.01-0.03 mg / mL, preferably 0.015-0.025 mg / mL; PAA-MoWS2 nanoparticles are prepared as an aqueous solution 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 affects the initial fluorescence emission intensity of F0, and the amount of MoWS2 affects the degree of quenching, which may in turn affect the response and signal-to-noise ratio. A certain concentration yields a better signal-to-noise ratio and responsiveness, resulting in superior sensitivity for Aspergillus flavus detection.

[0029] According to the above scheme, the method for constructing the standard curve of fluorescence emission intensity versus Aspergillus flavus spore concentration is as follows: Aspergillus flavus spore solutions of different concentrations are obtained using Aspergillus flavus toxin-producing standard strains, and different concentrations of spore solutions are inoculated into peanuts to achieve a final spore concentration of 10. 2 -10 7The concentration of Aspergillus flavus spores was 1 / g. After incubation, total DNA was extracted, amplified by PCR, and incubated with EuNPs-probeDNA solution. Then, an aqueous solution of MoWS2 nanoparticles was added, and the fluorescence emission intensity under 360nm excitation was measured. A standard curve of fluorescence recovery degree (F-F0) / F0 and Aspergillus flavus spore concentration was established by correlating the concentration of Aspergillus flavus spores with the fluorescence recovery degree.

[0030] The specific method is as follows: First, Aspergillus flavus standard strain 3.4408 was inoculated onto Czapek-Dox Agar (CDA) solid medium and cultured at 28℃ and 90% humidity for 7 days. The spores were then washed with 5% glycerol / water, and spore counting was performed using a hemocytometer. 10g of blank peanut sample was ground into peanut powder with a diameter less than 500μm using a laboratory grinder. Subsequently, 0.2g was accurately weighed and transferred to a 2.0mL test tube without nuclease. The obtained spore solution was then serially diluted 10-fold to 10... 2 -10 7 200 μL of spore solutions of different concentrations were inoculated into peanuts at a spore concentration of 10 spores / mL. 2 -10 7 The concentration of Aspergillus flavus spores was measured at 28°C for 1 hour. Total DNA was extracted using the CTAB method, amplified by PCR, and detected by a fluorescence sensor. The fluorescence emission intensity at 615 nm was measured using a fluorescence spectrophotometer under 360 nm excitation. A standard curve for detecting Aspergillus flavus in peanuts was established by correlating the concentration of Aspergillus flavus spores with the fluorescence recovery rate (F-F0) / F0.

[0031] This invention utilizes rare-earth europium fluorescent nanoparticles (EuNPs) as the energy donor and PAA-modified MoWS2 nanoparticles as the energy acceptor. Probe DNA is coupled with EuNPs to obtain EuNPs-probe DNA. The interaction between the DNA probe and the MoWS2 nanoparticles shortens their distance, promoting fluorescence resonance energy transfer (FRET). When the target DNA or the Aspergillus flavus nor-1 gene (in samples containing Aspergillus flavus) is present, it specifically hybridizes with the probe DNA to form double-stranded DNA, weakening the interaction between the probe DNA and the MoWS2 nanoparticles. This increases the distance between EuNPs and MoWS2 nanoparticles, inhibiting FRET and thus restoring the fluorescence of EuNPs. This establishes a fluorescence sensor based on a fluorescence recovery mechanism. It features strong binding to target molecules, high sensitivity, good selectivity, and suitability for detecting Aspergillus flavus in real samples. Rapid and highly sensitive detection of Aspergillus flavus is achieved by monitoring changes in fluorescence emission intensity (the correlation between the degree of fluorescence recovery and the concentration of target DNA or Aspergillus flavus spores).

[0032] The beneficial effects of this invention are:

[0033] The fluorescence resonance energy transfer sensor of this invention is a fluorescence sensor based on a fluorescence mechanism of fluorescence resonance energy transfer and target-specific recognition. It uses rare-earth europium fluorescent nanoparticles as the energy donor and PAA-modified MoWS2 nanoparticles as the energy acceptor. The interaction between the DNA probe and the MoWS2 nanoparticles shortens the distance between them, promoting fluorescence resonance energy transfer. Through specific complementary hybridization of the target DNA or the Aspergillus flavus nor-1 gene with the probe DNA, a DNA double helix structure is formed, weakening the interaction between the single-stranded DNA probe and the MoWS2 nanoparticles. This increases the distance between the energy donor and acceptor pairs, hindering the fluorescence resonance energy transfer process and causing the EuNPs fluorescence to recover. The change in fluorescence recovery intensity enables the detection of Aspergillus flavus. This method is simple to operate, rapid, and suitable for rapid detection and analysis.

[0034] This invention utilizes a one-step method to prepare MoWS2 nanoparticles with good water solubility. For the first time, it employs MoWS2 nanoparticles, which possess excellent fluorescence quenching ability and biocompatibility, as an energy donor. Combined with EuNPs, which exhibit high fluorescence quantum yield and good fluorescence performance, a fluorescence resonance energy transfer sensor is constructed based on the principles of fluorescence resonance energy transfer (FRET) and DNA complementary pairing. The Aspergillus flavus detection method provided by this invention is expected to provide key technical support for the source-based inhibition of aflatoxin and its toxin-producing fungi, and has broad application prospects in aflatoxin early warning and control research and the detection of other food contaminants. Attached Figure Description

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

[0036] Figure 2 Schematic diagram showing the overlap between the fluorescence emission spectra of EuNPs and the ultraviolet spectra 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 curves for fluorescence emission intensity at different concentrations of target DNA. F0 represents the fluorescence emission intensity in the absence of target DNA, and F represents the fluorescence emission intensity at different concentrations of target DNA.

[0039] Figure 5Standard curves for fluorescence emission intensity of Aspergillus flavus spores at different concentrations. F0 represents the fluorescence emission intensity when the Aspergillus flavus spore concentration is 0, and F represents the fluorescence emission intensity of spores at different concentrations of Aspergillus flavus. Detailed Implementation

[0040] To enhance understanding of the present invention, it will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are mainly for further illustrating the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps or conditions of the present invention without departing from the essence of the present invention are within the scope of the present invention.

[0041] Example 1

[0042] Construction of a fluorescence resonance energy transfer sensor:

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

[0044] The specific preparation process is as follows: styrene monomer (10 mM) and acrylic monomer (0.95 mM) are dissolved in 10 mL of deionized water containing 0.45 mM sodium dodecyl sulfonate (SDS), added to a round-bottom flask, and stirred evenly with a magnetic stir bar. Then, the air in the round-bottom flask is removed with high-purity nitrogen and heated to 70°C. Next, 0.5 mL of 0.15 mM potassium persulfate is added to the flask, and the reaction is stirred for 8 hours. After cooling to room temperature, the polystyrene nanoparticles with carboxyl groups are collected by filtration through Whatman 2V filter paper (8 μm pore size) and purified by dialysis using a dialysis bag (molecular weight cutoff 30000 Da). The polystyrene nanoparticles prepared above were diluted with 10 mL of acetone-water (v / v = 1:1), followed by the addition of 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). The mixture was stirred at 60 °C and reacted in the dark for 10 h, then cooled to room temperature and reacted for another 2 h. Finally, the organic solvent in the solution was removed by vacuum distillation, and the mixture was dialyzed with deionized water for 5 days to remove any remaining small molecules. The liquid in the dialysis bag was collected, and 0.05% sodium azide was added and stored at 4 °C.

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

[0046] Take 2 mg of EuNPs prepared in step 1 and sonicate them in 2 mL of morpholine ethanesulfonic acid (MES, pH = 5.5, 10 mM) buffer. Add 0.4 mM 1-ethyl-(3-dimethylaminopropyl)-carbonyldiimide hydrochloride (EDC·HCl) and 1 mM N-hydroxysuccinimide (NHS). After incubation at room temperature for 40 min, centrifuge to collect the precipitate and obtain EuNPs with carboxyl-activated surface. Add 2 nmol of probe DNA and react overnight at room temperature. Add 10 mg of tris(hydroxymethyl)aminomethane (Tris) to terminate the reaction and obtain the EuNPs-modified DNA probe, denoted as EuNPs-probe DNA. After centrifugation and washing with water, finally disperse EuNPs-probe in 2 mL of HEPES (pH = 7.4, 10 mM) buffer for later use.

[0047] The 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, MoWS2 nanoparticles were prepared. 3.85 mmol Na2MoO4·2H2O and 0.15 mmol Na2WO4·2H2O were dissolved in 40 mL of deionized water. Then, 15 mmol CH4N2S and 3 mmol C2H2O4·2H2O were added with constant stirring. After the precursor solution transformed into a transparent liquid, it was transferred to a 60 mL high-pressure reactor and heated to 200 °C for 12 h. After cooling to room temperature, the nanoparticles were washed three times with high-purity water and ethanol, respectively, and then dried in a vacuum drying oven. Next, polyacrylic acid (PAA) modified MoWS2 nanoparticles (PAA-MoWS2) were prepared. Weigh 20 mg of PAA and place it in a 100 mL round-bottom flask containing 20 mL of ultrapure water. Stir well, then add 20 mg of MoWS2 powder. Sonicate at room temperature for 12 h to obtain a black dispersion. Centrifuge at 3000 rpm for 10 min to remove large particles. Centrifuge the obtained supernatant and wash it with water three times (10000 rpm, 10 min). Dry it under vacuum at 60 °C and disperse it in high-purity water to a concentration of 1 mg / mL. Store at room temperature for later use.

[0050] Transmission electron microscopy characterization image of PAA-MoWS2 nanoparticles as shown below Figure 1 As shown in (A); PAA-MoWS2 aqueous dispersion as shown in Figure 1 As shown in (B), the schematic diagram illustrates the overlap between the fluorescence emission spectra of EuNPs and the UV spectra of MoWS2 nanoparticles. Figure 2 As shown.

[0051] 3) A method for constructing a MoWS2-based fluorescence resonance energy transfer sensor was used. 0.02 mg / mL EuNPs-probe DNA 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. With increasing PAA-MoWS2 concentration, the fluorescence quenching efficiency gradually increased. When the PAA-MoWS2 concentration was 0.02-0.04 mg / mL, the fluorescence quenching efficiency was above 70%, and at a PAA-MoWS2 concentration of 0.04 mg / mL, the fluorescence quenching efficiency reached 87%. Figure 3 As shown in (A), within this concentration range, the non-specific quenching caused by incubation of PAA-MoWS2 with EuNPs is almost negligible. Specifically, the fluorescence spectra before and after incubation of 0.02 mg / mL EuNPs with 0.04 mg / mL PAA-MoWS2 are shown in Figure [Figure Number]. Figure 3 As shown in (B). The MoWS2 nanoparticle concentration of 0.04 mg / mL was selected for the fluorescence recovery experiment.

[0052] Example 2: Target DNA Detection

[0053] Different concentrations of target DNA standard solutions (0, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 3 nM, and 5 nM, respectively) were incubated with 0.02 mg / mL EuNPs-probe DNA. Then, 0.04 mg / mL MoWS2 nanoparticles were added. The fluorescence emission intensity of the solution was measured at 615 nm using a fluorescence spectrophotometer, and a standard curve was established between F-F0 / F0 and the target DNA concentration. The fluorescence emission intensity measured after incubation of 0.02 mg / mL EuNPs-probe DNA with 0.04 mg / mL PAA-MoWS2 in the absence of target DNA was defined as F0. Figure 4 As shown.

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

[0055] Example 3: Detection of Aspergillus flavus in peanut samples:

[0056] The constructed fluorescent sensor was used to detect Aspergillus flavus in peanut samples. The detection procedure was as follows: Aspergillus flavus standard strain 3.4408 was inoculated onto Czapek-Dox Agar (CDA) solid medium and cultured at 28℃ and 90% humidity for 7 days. Spores were then washed with 5% glycerol / water, and spore counts were performed using a hemocytometer. 10g of blank peanut sample was ground into peanut powder with a diameter less than 500μm using a laboratory grinder. Subsequently, 0.2g was accurately weighed and transferred to a nuclease-free 2.0mL test tube. The obtained spore solution was serially diluted 10-fold to 10... 2 -10 7 200 μL of spore solutions of different concentrations were inoculated into peanuts at a spore concentration of 10 spores / mL. 2 -10 7 / g. After incubation at 28℃ for 1 h, total DNA was extracted using the CTAB method, amplified by PCR, and then the fluorescence emission intensity at 360 nm excitation was measured using a fluorescence spectrophotometer in the same manner as in Example 2. A standard curve for detecting Aspergillus flavus in peanuts was established by correlating the concentration of Aspergillus flavus spores with the degree of fluorescence recovery. The detection standard curve established with (F-F0) / F0 as the ordinate and the concentration of Aspergillus flavus spores as the abscissa is shown below. Figure 5 As shown.

[0057] F0 represents the fluorescence emission intensity when the concentration of Aspergillus flavus spores is 0, which 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. The fluorescence recovery degree (F - F0) / F0 has a good linear relationship with the number of Aspergillus flavus spores.

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

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

[0061] Recovery determination of sample spiking: 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 6The sample was incubated at 37°C for 1 hour after inoculation, followed by total DNA extraction and PCR amplification. The PCR amplification products were then detected using the aforementioned biosensor to determine the Aspergillus flavus content in the sample. The recovery rates are shown in Table 1. Analysis of the intra- and inter-group results showed that the recovery rate of Aspergillus flavus spores was 95-109%, with a coefficient of variation of 0.35-7.3%. The high recovery rate and low coefficient of variation indicate that this method has good reproducibility and accuracy in detecting Aspergillus flavus. This method, which detects aflatoxin-producing genes at the source, is suitable for rapid detection and early warning analysis.

[0062] Table 1 Results of spiked experiment for detecting Aspergillus flavus in peanuts using a fluorescence sensor.

[0063]

Claims

1. A fluorescent resonance energy transfer sensor based on MoWS2 nanoparticles, characterized in that: EuNPs-probe DNA formed by polyacrylic acid-MoWS2 nanoparticles and DNA probe modified EuNPs, the DNA probe is a probe specifically recognizing Aspergillus flavus nor-1, and the sequence of the DNA probe is 5'-CAACCCGCCTGATGCTGCAGCAGTC-3', The preparation method of the polyacrylic acid-MoWS2 nanoparticles is as follows: Preparation of MoWS2 nanoparticles: under continuous stirring, CH4N2S and C2H2O4·2H2O are added into a solution of sodium molybdate and sodium tungstate, and the reaction is heated at 200-220 °C for 10-14 h, and MoWS2 is obtained after post-processing; MoWS2 is added into a polyacrylic acid solution, and the mass ratio of polyacrylic acid to MoWS2 is 1:1-3, ultrasonic treatment is performed at room temperature to obtain a black dispersion liquid, large particles are removed by centrifugation at a speed of 3000-5000 rpm, the supernatant is centrifuged to obtain a precipitate, water is added, and the precipitate is collected by centrifugation at a speed of 10000-12000 rpm, vacuum drying, dispersion in water, and storage at room temperature for standby.

2. The fluorescence resonance energy transfer sensor of claim 1, wherein: The polyacrylic acid-MoWS2 nanoparticles are prepared into an aqueous solution for use; the DNA probe modified EuNPs are prepared into a solution for use; the DNA probe is modified with an amino group, the surface of the EuNPs is modified with a carboxyl group, the amino-modified DNA probe is coupled with the carboxyl-modified EuNPs to obtain EuNPs-probe DNA.

3. The fluorescence resonance energy transfer sensor of claim 1, wherein: The amount of the polyacrylic acid-MoWS2 nanoparticles is determined according to the requirement that the fluorescence emission intensity of the EuNPs-probe DNA is quenched by more than 70%.

4. The method of claim 1 for the preparation of MoWS2 nanoparticle based fluorescence resonance energy transfer sensor, characterized by: The method comprises the following steps: 1) providing EuNPs for standby; 2) preparation of MoWS2 nanoparticles: under continuous stirring, CH4N2S and C2H2O4·2H2O are added into a solution of sodium molybdate and sodium tungstate, and the reaction is heated at 200-220 °C for 10-14 h, and MoWS2 is obtained after post-processing; MoWS2 is added into a polyacrylic acid solution, and the mass ratio of polyacrylic acid to MoWS2 is 1:1-3, ultrasonic treatment is performed at room temperature to obtain a black dispersion liquid, large particles are removed by centrifugation at a speed of 3000-5000 rpm, the supernatant is centrifuged to obtain a precipitate, water is added, and the precipitate is collected by centrifugation at a speed of 10000-12000 rpm, vacuum drying, dispersion in water, and storage at room temperature for standby; 3) modification of probe DNA: providing surface carboxyl-activated EuNPs, modifying the probe DNA to obtain modified EuNPs-probe DNA, and the DNA probe is a probe specifically recognizing Aspergillus flavus nor-1, and the sequence of the DNA probe is 5'-CAACCCGCCTGATGCTGCAGCAGTC-3'.

5. The method of claim 4, comprising the steps of: The modification step of the probe DNA is as follows: the probe DNA is added into the surface carboxyl-activated EuNPs, and the reaction is performed overnight, and EuNPs-probe DNA is obtained after post-processing.

6. A rapid detection method of Aspergillus flavus based on the fluorescence resonance energy transfer sensor according to claim 1 for non-disease diagnosis and treatment purposes, characterized by: The DNA of Aspergillus flavus in the sample to be tested is extracted, and after PCR amplification of nor-1, incubation with the solution of EuNPs-probe DNA in the fluorescence resonance energy transfer sensor of claim 1, and addition of an aqueous solution of polyacrylic acid-MoWS2 nanoparticles, the fluorescence emission intensity under excitation at 360 nm is measured, and based on the standard curve of the fluorescence recovery degree (F-F0) / F0 and the concentration of Aspergillus flavus spores, F0: the initial fluorescence emission intensity without the sample to be tested, F: the fluorescence emission intensity measured in the presence of the sample to be tested, the concentration of Aspergillus flavus spores in the sample to be tested is analyzed.

7. The method for rapid detection of Aspergillus flavus according to claim 6, characterized in that: The EuNPs-probe DNA is dispersed in HEPES buffer for use, and the use concentration is 0.015-0.025 mg / mL; the polyacrylic acid-MoWS2 nanoparticles are prepared into an aqueous solution for use, and the use concentration is 0.025 mg / mL-0.04 mg / mL.

8. The method for rapid detection of Aspergillus flavus according to claim 6, characterized in that: The method for constructing the standard curve of the fluorescence emission intensity and the concentration of Aspergillus flavus spores is as follows: using Aspergillus flavus toxin-producing standard bacteria to obtain Aspergillus flavus spore solutions with different concentrations, inoculating the spore solutions with different concentrations into peanuts, so that the final concentration of spores is 10 2 -10 7 After incubation, total DNA is extracted, amplified by PCR, incubated with a solution of EuNPs-probe DNA, and then an aqueous solution of polyacrylic acid-MoWS2 nanoparticles is added. The fluorescence emission intensity under excitation at 360 nm is determined, and the standard curve of the fluorescence recovery degree, i.e. (F-F0) / F0, and the concentration of Aspergillus flavus spores is established by the correlation between the concentration of Aspergillus flavus spores and the fluorescence recovery degree.

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