Kit for rapidly detecting aflatoxin B1 based on catalytic characteristics of gold-platinum alloy nanoparticles and application of kit

Abstract

The invention discloses a kit for rapidly detecting aflatoxin B1 based on catalytic characteristics of gold-platinum alloy nanoparticles and application of the kit. The kit comprises a test strip, Apt / Au-Pt NPs detection probe dispersion liquid and a hydrogen peroxide aqueous solution, and the test strip comprises a bottom plate, a sample absorption pad, a nitrocellulose membrane and a water absorption pad; the nitrocellulose membrane is coated with an SA / Biotin-cDNA (complementary deoxyribonucleic acid) and an SA / Biotin-DNA1 (deoxyribonucleic acid 1); the Apt / Au-Pt NPs detection probe is formed by reducing a disulfide bond of SH-Apt by using tris (2-formylethyl) phosphine hydrochloride and then combining the SH-Apt with Au-Pt NPs through an Au-S bond. According to the kit, the aflatoxin B1 can be rapidly detected through the change of the air pressure value, and the kit has the advantages of being high in detection sensitivity, high in specificity, simple, convenient and accurate in quantification.

Description

Technical Field

[0001] The present invention belongs to the technical field of aflatoxin detection, and particularly relates to a kit for rapidly detecting aflatoxin B1. The kit utilizes an aptamer labeled with gold-platinum alloy nanoparticles (denoted as Apt / Au-Pt NPs detection probe), and combines with a handheld barometer to achieve portable and rapid detection of aflatoxin B1. Background Art

[0002] Mycotoxins are widely distributed in food crops such as grains. Approximately 25% of the world's food crops are contaminated by mycotoxins, resulting in a large amount of food crops being inedible, and food crops contaminated by mycotoxins seriously threaten people's lives. Mycotoxins mainly include toxins such as ochratoxin, aflatoxin, zearalenone, and vomitoxin. Among them, aflatoxin B1 has the strongest toxicity and carcinogenicity. Aflatoxin B1 is stable in nature, and conventional processing methods cannot completely remove aflatoxin B1 from food. Therefore, establishing a simple, rapid, and effective detection method for aflatoxin B1 is of great significance for ensuring food safety and human life safety.

[0003] Traditional methods for detecting aflatoxin B1 include: gas chromatography, liquid chromatography, liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay, etc. These detection methods have the advantages of high sensitivity and good accuracy. However, there are also problems such as the need for large-scale instruments, complex operations, high detection costs, and the need for professional operators, and they are not suitable for on-site rapid detection in large-scale production. Therefore, there is an urgent need to develop a method for portable, simple, and rapid detection of aflatoxin B1. Summary of the Invention

[0004] The object of the present invention is to provide a kit for rapidly detecting aflatoxin B1 based on the catalytic properties of gold-platinum alloy nanoparticles, and a method for rapidly detecting aflatoxin B1 using the kit.

[0005] The kit for rapidly detecting aflatoxin B1 based on the catalytic properties of gold-platinum alloy nanoparticles provided by the present invention includes a test strip, an Apt / Au-Pt NPs detection probe dispersion, and an aqueous hydrogen peroxide solution.

[0006] The above test strip includes a bottom plate, a sample absorption pad, a nitrocellulose membrane, and a water absorption pad that are sequentially fixed on the bottom plate. A detection area and a quality control area are provided on the nitrocellulose membrane. The detection area is coated with a complex of streptavidin (SA) and complementary nucleotide 1 of biotinylated aflatoxin B1 aptamer, and the quality control area is coated with a complex of streptavidin and complementary nucleotide 2 of biotinylated aflatoxin B1 aptamer; the complementary nucleotide 1 of biotinylated aflatoxin B1 aptamer is Biotin-cDNA, and the complementary nucleotide 2 of biotinylated aflatoxin B1 aptamer is Biotin-DNA1; the nucleotide sequence of Biotin-cDNA is: Biotin-AGACACAGAGAGACAA, and the nucleotide sequence of Biotin-DNA1 is: Biotin-AAAAA AAAAAA.

[0007] The above Apt / Au-Pt NPs detection probe dispersion is formed by dispersing the Apt / Au-Pt NPs detection probe in a Tris-HCl buffer solution; the Apt / Au-Pt NPs detection probe is formed by reducing the disulfide bond of thiolated aflatoxin B1 aptamer with tris(2-formylethyl)phosphine hydrochloride and then binding it to gold-platinum alloy nanoparticles (Au-Pt NPs) through an Au-S bond; the thiolated aflatoxin B1 aptamer is SH-Apt; the nucleotide sequence of SH-Apt is: SH-TTTTTTTTTTGGGCACGTGTTGTCTCTCTGTGTCTCGTG CCC. SH-Apt can bind to the above Biotin-cDNA and Biotin-DNA1 respectively.

[0008] Furthermore, in the above kit, it is preferred that the concentration of Apt in the Apt / Au-Pt NPs detection probe dispersion is 20-25 μM, and the concentration of Au-Pt NPs is 40-50 μg / mL.

[0009] The preparation method of the above Au-Pt NPs is as follows: Heat an aqueous solution of chloroauric acid with a volume concentration of 2%-3% to boiling under magnetic stirring, then add trisodium citrate, continuously stir until the solution turns wine red, and then naturally cool to room temperature. Add chloroplatinic acid, heat to 75-80 °C under magnetic stirring, then add ascorbic acid, stir for 25-30 minutes, and then naturally cool to room temperature. After centrifugal washing, gold-platinum alloy nanoparticles Au-Pt NPs are obtained and stored at 4-6 °C.

[0010] Furthermore, in the preparation method of the above Au-Pt NPs, it is preferred that the molar ratio of chloroauric acid to trisodium citrate, chloroplatinic acid, and ascorbic acid is 1:3-4:0.4-0.7:9-11.

[0011] Further, in the above-mentioned kit, it is preferred that the mass concentration of hydrogen peroxide in the aqueous hydrogen peroxide solution is 25% to 30%.

[0012] Further, in the above-mentioned kit, it is preferred that the nitrocellulose membrane is CN140.

[0013] The present invention also provides the application of the above-mentioned kit in detecting aflatoxin B1.

[0014] The method for detecting aflatoxin B1 (AFB1) using the kit of the present invention comprises the following steps:

[0015] Step 1: Add aqueous solutions of aflatoxin B1 with different concentrations to the Apt / Au-Pt NPs detection probe dispersion, incubate at room temperature for 5 to 10 minutes to obtain a test solution; drop the test solution onto the sample absorption pad of the test strip, and incubate at room temperature for 3 to 5 minutes; then cut off the detection area of the test strip, put it into a sealed bottle containing an aqueous hydrogen peroxide solution, incubate at room temperature for 20 to 25 minutes, use a barometer to record the change in air pressure in the bottle, and plot a standard curve with the logarithm of the aflatoxin B1 concentration as the abscissa and the change in air pressure in the bottle as the ordinate.

[0016] Step 2: Add the test sample to the Apt / Au-Pt NPs detection probe dispersion, incubate at room temperature for 5 to 10 minutes to obtain a sample solution; drop the sample solution onto the sample absorption pad of the test strip, and incubate at room temperature for 3 to 5 minutes; then cut off the detection area of the test strip, put it into a sealed bottle containing an aqueous hydrogen peroxide solution, incubate at room temperature for 20 to 25 minutes, use a barometer to record the change in air pressure in the bottle, and calculate the concentration of aflatoxin B1 in the test sample by combining with the standard curve established in Step 1.

[0017] The present invention improves the synthesis method of gold-platinum alloy nanoparticles to enhance their peroxidase-like activity, and then combines the thiol-modified aflatoxin B1 aptamer with the gold-platinum alloy nanoparticles through Au-S bonds to form an Apt / Au-Pt NPs detection probe. In the present invention, the detection zone and the quality control zone on the nitrocellulose membrane of the test strip are respectively coated with a complex of streptavidin and biotin-modified complementary DNA strands. The streptavidin and the biotin-modified complementary DNA strands are combined through the interaction between streptavidin and biotin to respectively prepare SA / Biotin-cDNA and SA / Biotin-DNA1, and the SA / Biotin-cDNA and SA / Biotin-DNA1 are respectively fixed on the detection zone and the quality control zone of the nitrocellulose membrane using a membrane scribing instrument. When the Apt / Au-Pt NPs detection probe is present, the detection probe is captured in the detection zone through the base complementary pairing binding of Apt and the complementary DNA strand. When aflatoxin B1 is present, aflatoxin B1 competes with the complementary DNA strand in the detection zone for binding to the detection probe, causing the detection probe to fall off from the detection zone, resulting in a reduction in the Apt / Au-Pt NPs detection probe on the detection zone. Since the gold-platinum alloy nanoparticles have peroxidase-like activity and can catalyze the decomposition of hydrogen peroxide to generate oxygen, the air pressure of the system changes. Therefore, a handheld barometer is used to detect the air pressure value of the system, and through the change in the air pressure value, rapid detection of aflatoxin B1 can be achieved.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. The present invention optimizes the synthesis method of gold-platinum alloy nanoparticles in the Apt / Au-Pt NPs detection probe, improves the peroxidase-like activity of the gold-platinum alloy nanoparticles, and enhances the analysis sensitivity and analysis efficiency of aflatoxin B1.

[0020] 2. In the kit of the present invention, aflatoxin B1 competes with the complementary DNA strand in the detection zone of the test strip for binding to the Apt / Au-Pt NPs detection probe, resulting in a reduction in the gold-platinum alloy nanoparticles in the detection zone. Utilizing the peroxidase-like activity of the gold-platinum alloy nanoparticles, the air pressure of the experimental system changes, and accurate quantification of aflatoxin B1 can be achieved using only a barometer without complex precision instruments.

[0021] 3. The kit of the present invention has the advantages of high sensitivity, strong specificity, simplicity and convenience, and accurate quantification for detecting aflatoxin B1. Brief Description of the Drawings

[0022] Figure 1 It is a schematic diagram of the principle of the method for visual detection of aflatoxin B1.

[0023] Figure 2 They are the transmission electron microscope image of Au-Pt NPs (A) and the particle size distribution histogram of Au-Pt NPs (B).

[0024] Figure 3 It is the elemental distribution map of Au-Pt NPs powder.

[0025] Figure 4 They are the total X-ray photoelectron spectroscopy map of Au-Pt NPs powder (A), the Au 4f map of X-ray photoelectron spectroscopy of Au-Pt NPs powder (B), and the Pt 4f map of X-ray photoelectron spectroscopy of Au-Pt NPs powder (C).

[0026] Figure 5 It is the graph of air pressure change after different nanomaterials catalyze hydrogen peroxide.

[0027] Figure 6 It is the graph of air pressure change after different Au-Pt NPs catalyze hydrogen peroxide.

[0028] Figure 7 It is the ultraviolet absorption of Apt / Au-Pt NPs detection probe dispersion liquid and pure water.

[0029] Figure 8 They are the air pressure change of the system in the presence of different concentrations of AFB1 (A) and the linear relationship between ΔP and AFB1 concentration in the range of 1 - 500 ng / mL (B).

[0030] Figure 9 It is the response of the system in the presence of different mycotoxins. Detailed implementation manners

[0031] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited to these embodiments only.

[0032] The detection principle of the present invention is as Figure 1As shown, a mixed solution of the sample and the Apt / Au-Pt NPs detection probe dispersion is dropped onto the sample absorption pad. When there is no aflatoxin B1 in the sample, as the test solution flows to the detection area of the nitrocellulose membrane, the Biotin-cDNA in the detection area binds to the Apt base on the Apt / Au-Pt NPs detection probe through complementary base pairing, thereby fixing the Apt / Au-Pt NPs detection probe in the detection area. When the test solution flows to the quality control area of the nitrocellulose membrane, the Biotin-DNA1 in the quality control area binds to the Apt base on the remaining Apt / Au-Pt NPs detection probe through complementary base pairing, thereby fixing the Apt / Au-Pt NPs detection probe in the quality control area. Since Au-Pt NPs have peroxidase-like activity, they can catalyze hydrogen peroxide to produce oxygen. Therefore, after cutting out the detection area or the quality control area and placing it in a sealed bottle containing an aqueous hydrogen peroxide solution, as time goes by, the air pressure in the bottle gradually increases. When there is aflatoxin B1 in the sample, aflatoxin B1 competes with the Biotin-cDNA in the detection area of the nitrocellulose membrane to bind to the Apt / Au-Pt NPs detection probe. Since the binding ability of aflatoxin B1 to the Apt / Au-Pt NPs detection probe is stronger, the more aflatoxin B1 there is, the fewer Apt / Au-Pt NPs detection probes bound to the Biotin-cDNA in the detection area. The binding of Biotin-DNA1 in the quality control area to the Apt / Au-Pt NPs detection probe is not affected by aflatoxin B1. Therefore, when the detection area is cut out and placed in a sealed bottle containing an aqueous hydrogen peroxide solution with a mass concentration of 25% - 30%, within the same time, the change in air pressure in the bottle decreases with the increase of aflatoxin B1 in the sample, while the air pressure in the bottle with the quality control area added remains stable and does not change with the change in the content of aflatoxin B1. Therefore, the content of aflatoxin B1 in the sample is detected by the change in air pressure in the bottle with the detection area added.

[0033] Example 1

[0034] The kit for detecting aflatoxin B1 in this example includes a test strip, an Apt / Au-Pt NPs detection probe dispersion, and an aqueous hydrogen peroxide solution with a mass concentration of 30%.

[0035] The test strip includes a bottom plate and a sample absorption pad, a nitrocellulose membrane, and a water absorption pad that are sequentially pasted and fixed on the bottom plate. The nitrocellulose membrane is CN140, and a detection area and a quality control area are provided on the nitrocellulose membrane.

[0036] The detection area is coated with a complex of streptavidin (SA) and the complementary nucleotide 1 of biotinylated aflatoxin B1 aptamer. The biotinylated aflatoxin B1 aptamer complementary nucleotide 1 is Biotin-cDNA, and the nucleotide sequence of Biotin-cDNA is: Biotin-AGACACAGAGAGACAA. The specific preparation method of the detection area is as follows: Mix 300 μL of 5 μM biotinylated aflatoxin B1 aptamer complementary nucleotide 1, 120 μL of 50 μg / mL SA, and 180 μL of 10 mM PBS buffer, incubate at 37 °C for 1 h to obtain the SA-Biotin-cDNA complex; then use a HM3035 membrane scribing instrument to fix the SA-Biotin-cDNA complex on the detection area of the nitrocellulose membrane at a speed of 2 μL / cm.

[0037] The quality control area is coated with a complex of streptavidin and the complementary nucleotide 2 of biotinylated aflatoxin B1 aptamer. The biotinylated aflatoxin B1 aptamer complementary nucleotide 2 is Biotin-DNA1, and the nucleotide sequence of Biotin-DNA1 is: Biotin-AAAAAAAAAAA. The specific preparation method of the quality control area is as follows: Mix 300 μL of 5 μM biotinylated aflatoxin B1 aptamer complementary nucleotide 2, 120 μL of 50 μg / mL SA, and 180 μL of 10 mM PBS buffer, incubate at 37 °C for 1 h to obtain the SA-Biotin-DNA1 complex; then use a HM3035 membrane scribing instrument to fix the SA-Biotin-DNA1 complex on the quality control area of the nitrocellulose membrane at a speed of 2 μL / cm.

[0038] The Apt / Au-Pt NPs detection probe dispersion is formed by dispersing the Apt / Au-Pt NPs detection probe in Tris-HCl buffer; the Apt / Au-Pt NPs detection probe is formed by reducing the disulfide bond of the thiolated aflatoxin B1 aptamer with tris(2-carboxyethyl)phosphine hydrochloride and binding it to gold-platinum alloy nanoparticles Au-Pt NPs through Au-S bonds; the thiolated aflatoxin B1 aptamer is SH-Apt; the nucleotide sequence of SH-Apt is: SH-TTTTTTTTTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCC. The preparation method of the Apt / Au-Pt NPs detection probe dispersion includes the following steps:

[0039] Step 1: Prepare the Au-Pt NPs dispersion

[0040] Add 2 mL of 25 mM aqueous chloroauric acid trihydrate solution to 193 mL of water, heat to boiling under magnetic stirring, then add 5 mL of 39 mM aqueous sodium citrate solution. After continuous stirring for 45 min, the solution turns wine red. Let it cool naturally to room temperature, and after centrifugation and washing, obtain a gold nanoparticle (Au NPs) solution. Take the above Au NPs solution, add 25 mL of 1 mM aqueous chloroplatinic acid solution, heat to 80 °C under magnetic stirring, then add 50 mL of 10 mM aqueous ascorbic acid solution at a rate of 5 mL / min. After stirring for 30 min, let it cool naturally to room temperature, and after centrifugation and washing, obtain an Au-Pt NPs dispersion, and store it at 4 °C.

[0041] Step 2: Prepare the Apt / Au-Pt NPs detection probe dispersion

[0042] Mix 100 μL of SH-Apt with 5 μL of 2.0 mM aqueous solution of tris(2-formylethyl)phosphine hydrochloride for 30 min, then drop the resulting mixture into 1 mL of Au-Pt NPs dispersion, and incubate for 12 h under dark conditions to allow SH-Apt to bind to Au-Pt NPs through Au-S bonds; finally, centrifuge and separate the mixture and disperse it in Tris-HCl buffer to obtain the Apt / Au-Pt NPs detection probe dispersion, and store it at 4 °C. The concentration of Apt in the Apt / Au-Pt NPs detection probe dispersion is 24 μM, and the concentration of Au-Pt NPs is 45 μg / mL.

[0043] The morphology of the above Au-Pt NPs was characterized by high-resolution transmission electron microscopy, and the results are as Figure 2 shown in A. The Au-Pt NPs are round particles with uniform distribution and uniform particle size. Statistical analysis of their particle size showed that the size is between 12 and 17 nm, and it basically follows a normal distribution ( Figure 2 B), with an average size of 14.5 ± 0.5 nm. The elemental distribution of Au-Pt NPs was analyzed by X-ray energy dispersive spectrometer, and the results Figure 3 are shown Figure 3 in A for the distribution of Au-Pt NPs, Figure 3 B and Figure 3 D for the distribution of Au element and Pt element respectively. The Au element and Pt element are distributed on the whole nanoparticle and evenly distributed. Figure 3 C is the composite diagram obtained by superimposing Figure 3 A, B and D, Figure 3The yellow color in C is the composite color of green (Au element) and red (Pt element), indicating that both Au and Pt elements are distributed in the nanoparticles and there is no situation where the Au or Pt element is more widely distributed, proving that the synthesized nanoparticles are Au-Pt alloy nanoparticles (Au-Pt NPs). The elemental composition and the existing state of the elements of the synthesized material were further characterized by X-ray photoelectron spectroscopy, such as Figure 4 shown in A. The elemental characteristic peaks at 532.2 eV, 286.4 eV, 84.1 eV, and 73.8 eV in the XPS full spectrum of Au-Pt NPs are attributed to O 1s, C 1s, Au 4f, and Pt 4f, respectively. The Au 4f spectrum was deconvoluted and fitted ( Figure 4 B). The peaks at the binding energies of 84.1 eV and 87.6 eV correspond to the characteristic peaks of elemental Au, further proving the existence of Au. In the deconvoluted and fitted spectrum of Pt 4f ( Figure 4 C), the peaks at 71.1 eV and 74.3 eV belong to the characteristic peaks of elemental Pt, indicating the existence of Pt. Au-Pt NPs have peroxidase-like activity, that is, Au-Pt NPs can catalyze hydrogen peroxide to produce oxygen. Therefore, the peroxidase-like activity of the synthesized Au-Pt NPs was verified, such as Figure 5 shown. Au NPs, Pt NPs, and Au-Pt NPs all have peroxidase-like activity. However, under the same conditions, the peroxidase-like activity of Au-Pt NPs is stronger. The amount of oxygen produced by Au-Pt NPs catalyzing hydrogen peroxide in the same time is much higher than that of Au NPs and Pt NPs. Therefore, Au-Pt NPs with stronger peroxidase-like activity were selected to obtain higher detection performance. However, the synthesis method of Au-Pt NPs has a certain influence on the peroxidase-like activity of Au-Pt NPs, such as Figure 6 shown. Among them, Au-Pt NPs-1 are nanoparticles synthesized by the traditional one-step synthesis method, and Au-Pt NPs-2 are synthesized by the method provided in this example. Compared with the traditional method, the Au-Pt NPs synthesized by the method of the present invention are spherical particles with uniform particle size and have stronger peroxidase-like activity. In addition, the ultraviolet spectrum of Apt / Au-Pt NPs was scanned in the wavelength range of 200-500 nm, and the results are as Figure 7 shown. Only when the aptamer strand (Apt) exists, that is, when the Apt / Au-Pt NPs detection probe is successfully prepared, there is an obvious ultraviolet absorption peak at 260 nm, which is caused by the conjugated double bond system of purine rings and pyrimidine rings in the aptamer strand absorbing ultraviolet light.

[0044] Example 2

[0045] Application of the kit of Example 1 in the portable rapid detection of AFB1, and the specific method is as follows:

[0046] Step 1: Plot the standard curve

[0047] Add aqueous solutions of aflatoxin B1 with different concentrations to 1 mL of the Apt / Au-Pt NPs detection probe dispersion, incubate at room temperature for 5 min to obtain the solution to be measured; drop the solution to be measured on the sample absorption pad of the test strip, and incubate at room temperature for 5 min. Subsequently, cut off the detection area of the test strip and put it into a sealed bottle containing 400 μL of hydrogen peroxide aqueous solution with a mass concentration of 30%, incubate for 20 min, and use an HT-1895 type barometer to record the change in air pressure in the bottle. Plot the standard curve with the logarithm of the aflatoxin B1 concentration as the abscissa and the change in air pressure in the bottle as the ordinate.

[0048] From Figure 8 A and Figure 8 B, it can be seen that in the concentration range of 1 - 500 ng / mL, as the AFB1 concentration increases, the air pressure value in the bottle gradually decreases, and there is a good linear relationship between the AFB1 concentration and the air pressure change value (ΔP). The linear equation is ΔP = 17.455lg(C AFB1 ) + 3.110, and the correlation coefficient (R 2 ) is 0.997. After calculation, the lowest detection limit is (LOD = 3σ / K) 0.25 ng / mL.

[0049] Step 2: Detect AFB1 in the sample

[0050] Add the sample to be measured to the Apt / Au-Pt NPs detection probe dispersion, incubate at room temperature for 5 minutes to obtain the sample solution; drop the sample solution on the sample absorption pad of the test strip, and incubate at room temperature for 3 minutes; then cut off the detection area of the test strip and put it into a sealed bottle containing hydrogen peroxide aqueous solution, incubate at room temperature for 20 minutes, use a barometer to record the change in air pressure in the bottle, and calculate the concentration of aflatoxin B1 in the sample to be measured in combination with the standard curve established in Step 1. The specific experiment is as follows:

[0051] Use a high-speed grinder to crush peanut and corn samples, pass through a 32-mesh sieve, then take 5.00 g of the samples and place them in 50 mL centrifuge tubes respectively. Add 20 mL of a mixed solution of methanol and water with a volume ratio of 7:3, vortex and mix well, place in a shaker and oscillate for extraction for 20 min, centrifuge at 6000 r / min for 10 min, take the supernatant, and use the standard addition method to evaluate the performance of this method in the detection of AFB1 in actual samples, and compare the detection results of this method with those of the national standard method (high performance liquid chromatography), as shown in Table 1.

[0052] Table 1 Detection results of AFB1 in samples by the present method and high performance liquid chromatography

[0053]

[0054] As can be seen from Table 1, the recovery rate of the method of the present invention is 92.2% - 104.8% (relative standard deviation (RSD) is 1.1% - 4.9%); the recovery rate of high performance liquid chromatography is 96.9% - 105.3% (relative standard deviation (RSD) is 1.1% - 3.7%). The detection results of the method of the present invention are relatively consistent with those of high performance liquid chromatography, indicating that the detection method of the present invention has high accuracy.

[0055] To further prove the specific detection of AFB1 by the kit of the present invention, other mycotoxins were selected as the control group, and the concentration of each control group of mycotoxins was ten times that of the AFB1 group. The specific experiment is as follows:

[0056] Add 10 μL of aqueous solutions of different kinds of mycotoxins (ochratoxin A (OTA), zearalenone (ZEN), deoxynivalenol (DON), and aflatoxin B2 (AFB2) group with a concentration of 500 ng / mL, AFB1 group with a concentration of 50 ng / mL) to 1 mL of Apt / Au-PtNPs detection probe dispersion, incubate at room temperature for 5 min to obtain the solution to be tested, drop the solution to be tested on the sample pad of the test strip, and incubate at room temperature for 5 min. Subsequently, cut the detection area of the test strip, put it into a sealed bottle containing 400 μL of hydrogen peroxide aqueous solution with a mass concentration of 30%, incubate for 20 min, and use an HT-1895 type barometer to record the change in air pressure in the bottle.

[0057] From Figure 9 it can be seen that the signal value of the AFB1 group is much larger than those of the OTA, ZEN, DON, and AFB2 groups, and there are significant differences, indicating that other toxins have no interference on this method, and it shows that the kit of the present invention has good selectivity for detecting AFB1.

Claims

1. A kit for rapid detection of aflatoxin B1 based on the catalytic properties of gold-platinum alloy nanoparticles, characterized in that: The kit comprises a test strip, an Apt / Au-Pt NPs detection probe dispersion, and an aqueous hydrogen peroxide solution; The test strip comprises a bottom plate and a sample absorption pad, a nitrocellulose membrane and a water absorption pad fixed on the bottom plate in sequence; a detection area and a quality control area are arranged on the nitrocellulose membrane, the detection area is coated with a complex of streptavidin and biotinylated aflatoxin B1 aptamer complementary nucleotide 1, and the quality control area is coated with a complex of streptavidin and biotinylated aflatoxin B1 aptamer complementary nucleotide 2; the biotinylated aflatoxin B1 aptamer complementary nucleotide 1 is Biotin-cDNA, and the biotinylated aflatoxin B1 aptamer complementary nucleotide 2 is Biotin-DNA1; the nucleotide sequence of the Biotin-cDNA is: Biotin-AGA CACAGAGAGACAA, and the nucleotide sequence of the Biotin-DNA1 is: Biotin-AAAAAAAAA AA; The Apt / Au-Pt NPs detection probe dispersion is formed by dispersing the Apt / Au-Pt NPs detection probe in Tris-HCl buffer; the Apt / Au-Pt NPs detection probe is formed by reducing the disulfide bond of the thiolated aflatoxin B1 aptamer with tri(2-formylethyl)phosphine hydrochloride and then combining it with gold-platinum alloy nanoparticles Au-Pt NPs through Au-S bonds; the thiolated aflatoxin B1 aptamer is SH-Apt; the nucleotide sequence of SH-Apt is: SH-TTTTTTTTTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCC.

2. The kit for rapid detection of aflatoxin B1 based on the catalytic properties of gold-platinum alloy nanoparticles according to claim 1, characterized in that: The concentration of Apt in the Apt / Au-Pt NPs detection probe dispersion is 20-25 μM, and the concentration of Au-Pt NPs is 40-50 μg / mL.

3. The kit for rapid detection of aflatoxin B1 based on the catalytic properties of gold-platinum alloy nanoparticles according to claim 1, characterized in that: The gold-platinum alloy nanoparticles Au-Pt NPs are prepared by the following method: The chloroauric acid aqueous solution was heated to boiling under magnetic stirring, and then trisodium citrate was added, and stirring was continued until the solution turned wine red, and then naturally cooled to room temperature, chloroplatinic acid was added, and heated to 75-80°C under magnetic stirring, and then ascorbic acid was added. After stirring for 25-30 minutes, it was naturally cooled to room temperature, and after centrifugation and washing, gold-platinum alloy nanoparticles Au-Pt NPs were obtained, which were stored at 4-6°C.

4. The kit for rapid detection of aflatoxin B1 based on the catalytic properties of gold-platinum alloy nanoparticles according to claim 3, characterized in that: The molar ratio of the chloroauric acid to trisodium citrate, chloroplatinic acid and ascorbic acid is 1:3-4:0.4-0.7:9-11.

5. The kit for rapid detection of aflatoxin B1 based on the catalytic properties of gold-platinum alloy nanoparticles according to claim 1, characterized in that: The mass concentration of hydrogen peroxide in the aqueous hydrogen peroxide solution is 25% to 30%.

6. The kit for rapid detection of aflatoxin B1 based on the catalytic properties of gold-platinum alloy nanoparticles according to claim 1, characterized in that: The nitrocellulose membrane is CN140.

7. Use of the kit according to any one of claims 1 to 6 in detecting aflatoxin B1.

8. The method for detecting aflatoxin B1 using a kit according to any one of claims 1 to 6, characterized in that: The steps include: Step 1: Add different concentrations of aflatoxin B1 aqueous solution to the Apt / Au-Pt NPs detection probe dispersion, incubate at room temperature for 5 to 10 minutes to obtain a test solution; drip the test solution onto the sample absorption pad of the test strip, and incubate at room temperature for 3 to 5 minutes; then cut off the detection area of ​​the test strip, put it into a sealed bottle filled with hydrogen peroxide aqueous solution, incubate at room temperature for 20 to 25 minutes, use a barometer to record the change in air pressure in the bottle, and draw a standard curve with the logarithm of the aflatoxin B1 concentration as the horizontal axis and the change in air pressure in the bottle as the vertical axis; Step 2: Add the sample to be tested to the Apt / Au-Pt NPs detection probe dispersion and incubate at room temperature for 5 to 10 minutes to obtain a sample solution; drop the sample solution onto the sample absorption pad of the test strip and incubate at room temperature for 3 to 5 minutes; then cut off the detection area of ​​the test strip and place it in a sealed bottle containing an aqueous hydrogen peroxide solution, incubate at room temperature for 20 to 25 minutes, use a barometer to record the change in air pressure in the bottle, and calculate the concentration of aflatoxin B1 in the sample to be tested based on the standard curve prepared in step 1.

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