Preparation method and application of aflatoxin M1 aptamer test strip based on gold nanospheres

Abstract

The invention discloses a preparation method and application of an aflatoxin M1 aptamer test strip based on gold nanospheres, and belongs to the field of food safety detection. The preparation method of the aflatoxin M1 aptamer transverse flow test strip based on AuNPs is constructed by utilizing the characteristics of simplicity and convenience in operation, high detection speed and portability of an aptamer and adopting a method of competition of the aptamer and AFM1 at the same time. The method can be applied to food safety detection, and has the advantages of high detection speed, stable batch and the like on the basis of ensuring the sensitivity.

Description

Technical Field

[0001] The invention relates to a preparation method and application of an aflatoxin M1 aptamer test strip based on gold nanospheres, and belongs to the field of food safety detection. Background Art

[0002] Aflatoxin M1 (AFM1) is a hydroxylated derivative of aflatoxin B1 (AFB1). It is a toxic metabolic product produced by hydroxylation in the body of a type of animal after ingesting feed and food contaminated with AFB1. The World Health Organization (WHO) has classified it as a Class IIB carcinogen. After entering the human body or other mammals, AFM1 significantly inhibits the normal synthesis of liver tissue protein, leading to liver poisoning, manifested as damage to liver tissue; it is carcinogenic, teratogenic, and mutagenic, causing a variety of liver diseases, and further inducing liver cancer. Its toxicity is nearly 70 times that of arsenic. AFM1 has a relatively high stability during milk pasteurization or other heat treatment processes, and its pyrolysis temperature is as high as 300°C. Once formed, it is difficult to eliminate. In order to ensure food safety, countries around the world have established strict limit standards for AFM1 content.

[0003] At present, the methods for detecting AFM1 residues include high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography. Although these methods have high accuracy and sensitivity, they all require professionals to operate large instruments, and the samples also require complex pretreatment and high detection costs. Therefore, they are not suitable for fast, simple and cost-effective food safety testing.

[0004] Compared with antibodies, aptamers have the advantages of low synthesis cost, high efficiency, high stability, easy directional fixation, etc., and have high affinity and high specificity for the target. Compared with traditional detection methods (such as HPLC, ELISA, etc.), aptamer-based lateral flow test strips do not require special instruments or complex pre-treatment in detection, the overall time is short, the operation is simple and easy to learn, and the demand for detection personnel is small and low. It has the advantages of low cost, simple operation, fast detection speed, and high sensitivity. It can become an effective analytical method for detecting fungal toxins, especially suitable for rapid on-site detection and large-scale screening. However, the existing lateral flow test strips for detecting AFM1 use antibodies as recognition elements, which have poor stability and high synthesis cost. There is a lack of stable, highly sensitive, and low-cost aptamer lateral flow test strips for AFM1. Summary of the invention

[0005] In order to solve the above technical problems, the present invention aims to provide a method for preparing and applying an aflatoxin M1 aptamer lateral flow test strip based on AuNPs.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides an AuNPs aptamer conjugate (AuNPs@SH-polyA-A) that specifically recognizes aflatoxin M1. 21 ), is to couple gold nanoparticles AuNPs with a probe containing an aptamer; the probe containing an aptamer has 5'-ACTGCTAGAGATTTTCCACATAAAAAAAAAAAAAAAAAA (SEQ ID NO. 1)-SH-3'.

[0008] In one embodiment, the AuNPs aptamer conjugate AuNPs@SH-polyA-A 21 The method is prepared by incubating concentrated AuNPs and aptamer solution in the dark for a period of time, adding NaCl solution and continuing to incubate in the dark for a period of time, centrifuging and resuspending the precipitate with a resuspension solution.

[0009] In one embodiment, the preparation process is specifically as follows: take 1 mL of concentrated AuNPs, add a 100 μM activated aptamer solution, incubate at 4°C in the dark for 24 hours, add 90 μL of 1 M NaCl solution, continue incubating at 4°C in the dark for 24 hours, then centrifuge at 10,000 rpm for 20 minutes, add a resuspension solution to resuspend, and store at 4°C for later use.

[0010] In one embodiment, the preparation method of gold nanoparticles AuNPs is specifically as follows: take 99mL of ultrapure water in a conical flask, add 1mL, 1% chloroauric acid (HAuCl4), stir and heat the prepared solution to boiling, then add an appropriate amount of 1% trisodium citrate, boil for half an hour after the solution turns red, finally stir and cool to room temperature, centrifuge at 10000rpm for 10min, and resuspend with 0.05% Tween-20 to prepare.

[0011] In one embodiment, the resuspension solution is a mixed solution containing 1% polyethylene glycol, 0.02% MgSO4, 2.5% sucrose, 0.25% Tween-20, and 5% BSA, and the mixture is diluted with 0.01 mol / L PBS buffer solution.

[0012] The present invention provides an aflatoxin M1 aptamer test strip based on AuNPs, comprising an AuNPs aptamer conjugate AuNPs@SH-polyA-A 21 , an aptamer complementary chain and a streptavidin complex; the aptamer complementary chain and the streptavidin complex include SA-Bio-DNA T and SA-Bio-DNA C ; the SH-polyA-A 21 It has the sequence shown as 5'-ACTGCTAGAGATTTTCCACATAAAAAAAAAAAAAAAAAA-SH-3'.

[0013] In one embodiment, the aptamer complementary strand DNA T The nucleic acid sequence is shown as: 5'-Biotin-ATGTGGAAAATCTCTAGCAGTAAA (SEQ ID NO. 2)-3'.

[0014] In one embodiment, the aptamer complementary strand DNA C The nucleic acid sequence is shown as: 5'-Biotin-TTTTTTTTTTTTTTTTTT (SEQ ID NO. 3)-3'.

[0015] The present invention also provides a method for preparing an aflatoxin M1 aptamer test strip based on AuNPs, comprising:

[0016] (a) Preparation of the AuNPs aptamer conjugate AuNPs@SH-polyA-A 21 ;

[0017] (b) Preparation of aptamer complementary strand and streptavidin complex (SA-Bio-DNA T and SA-Bio-DNA C ):

[0018] Streptavidin at a concentration of 1 mg / mL was mixed with complementary DNA at a concentration of 100 μM. T and DNA C The mixture was mixed with PBS solution and incubated at 4°C for more than 2 hours. Then, the mixture was centrifuged at 6000 rpm for 20 minutes using a 30 kDA ultrafiltration tube. The mixture was centrifuged twice in total. The inner tube was inverted and centrifuged at 1800 rpm for 8 minutes to collect the liquid and prepare SA-Bio-DNA. T and SA-Bio-DNA C , stored at 4℃ for future use;

[0019] (c) Preparation of T-line and C-line:

[0020] The prepared SA-Bio-DNAT and SA-Bio-DNAC were sprayed onto nitrocellulose membrane (NC membrane) using a film gold spraying instrument to form T lines and C lines, and then dried in an oven for more than 2 h;

[0021] (d) Assembling aptamer test strips:

[0022] The sample pad was soaked in the sample pad treatment solution for more than 1 hour, dried at 37°C, and then the sample pad, NC membrane, and absorbent pad were assembled and fixed on the polyethylene base plate, cut into 3.5 mm wide pieces, and sealed and dried for storage.

[0023] In one embodiment, the complementary strand DNA in step (b) T The specific nucleic acid sequence is: 5'-Biotin-ATGTGGAAAATCTCTAGCAGTAAA-3'.

[0024] In one embodiment, the complementary strand DNA in step (b) C The specific nucleic acid sequence is: 5'-Biotin-TTTTTTTTTTTTTTTTTT-3'.

[0025] In one embodiment, the sample pad treatment solution in step (d) is a mixed solution containing 0.1% Tween-20, 1% BSA, and 2% sucrose, and the mixture is diluted with 0.01 mol / L PBS buffer solution.

[0026] The present invention also provides a method for detecting the content of aflatoxin M1 using the test strip, wherein the test sample is conjugated with the AuNPs aptamer conjugate AuNPs@SH-polyA-A 21 The mixture is mixed in a binding buffer containing HEPES, incubated at 36-38° C. for 45-90 min, and dripped onto the test strip. The aflatoxin M1 content is calculated based on the gray values ​​of the T line and the C line.

[0027] In one embodiment, the method is to combine the sample to be tested with the AuNPs aptamer conjugate AuNPs@SH-polyA-A 21 The mixture was mixed in a binding buffer containing HEPES, incubated at 37° C. for 45 min, and dripped onto the test strip. The aflatoxin M1 content was calculated based on the gray values ​​of the T line and the C line.

[0028] In one embodiment, the pH of the mixed solution is 6.0±0.2.

[0029] In one embodiment, the method is to take a photo of the color development of the test strip, measure the grayscale values ​​of the T line and the C line using the software Image J to obtain the T / C value, and calculate the aflatoxin M1 content based on the linear relationship between the T / C value and the AFM1 concentration.

[0030] The present invention also provides the test strip and application of the test strip in AFM1 detection.

[0031] In one embodiment, the application includes but is not limited to detecting aflatoxin M1 in food.

[0032] Beneficial effects:

[0033] (1) The present invention provides a method for preparing and applying an AuNPs-based aflatoxin M1 aptamer test strip, which can be photographed by a portable device (mobile phone) and then quantitatively detected by Image J software.

[0034] (2) The aptamer lateral flow test strip for detecting AFM1 prepared by the present invention is fast and time-saving, with a detection time of less than 15 minutes, and the detection batches are stable and the data repeatability is high. The relative colorimetric signal intensity (T / C) of the test strip shows a good linear relationship with the concentration of AFM1 in the concentration range of 2-500 ng / mL, and R 2 =0.9901. The test strip has high sensitivity, with a limit of detection (LOD) as low as 0.26ng / mL, which is lower than the antibody test strip (Sensitive competitive direct enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip for detecting aflatoxin M1 in milk 0.5-1.0ng / mL) and meets the requirements of the national standard GB / T 2761-2017 (limit 0.5ng / mL). BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the schematic diagram of the test strip.

[0036] Figure 2 This is the transmission electron microscopy (TEM) characterization image of AuNPs.

[0037] Figure 3 UV characterization pictures of AuNPs and AuNPs-aptamer.

[0038] Figure 4This is a graph showing the test results of the test strips testing AFM1 standard solutions of different concentrations.

[0039] Figure 5 This is the standard curve diagram for the test strip to detect AFM1.

[0040] Figure 6 It is the test strip specific detection result.

[0041] Figure 7 This is the T / C diagram for the specific detection of the test strip. DETAILED DESCRIPTION

[0042] Example 1 Preparation of aflatoxin M1 (AFM1) aptamer lateral flow test strip based on gold nanospheres

[0043] (1) Preparation of detection probe AuNPs@SH-polyA-A 21 :

[0044] AuNPs: Take 99mL ultrapure water in a conical flask, add 1mL of 1% HAuCl4, stir and heat the prepared solution to boiling, immediately add 2.7mL of 1% trisodium citrate, the solution gradually changes from colorless to purple, and finally to red. After the solution turns red, continue to stir and boil for 0.5h, finally stir and cool to room temperature, take 5mL of AuNPs solution and centrifuge at 10000rpm for 10min, remove the supernatant, and resuspend to 1mL with 0.05% Tween-20 as a protective agent, and store at 4℃ for later use;

[0045] AuNPs@SH-polyA-A 21 :Take 5μL SH-polyA-A 21 (100 μM), incubated at 95 °C for 5 min, cooled for 30 min, added 30 μL of 0.02 M trichloroethyl phosphate (TCEP) solution, activated at room temperature for 1 h, and activated SH-polyA-A 21 The solution was added to 1 mL of concentrated AuNPs, incubated in the dark at 4°C for 24 h, then 90 μL of 1 M NaCl solution was added and incubated in the dark for another 24 h, then centrifuged at 10,000 rpm for 20 min, the supernatant was removed, and a resuspension solution was added to resuspend to 1 mL, and stored at 4°C for later use; the resuspension solution was a mixed solution containing 1% polyethylene, 0.02% MgSO4, 2.5% sucrose, 0.25% Tween-20, and 5% BSA, and the above components were mixed in a 0.01 mol / L PBS buffer solution at a final concentration (v / v).

[0046] The SH-polyA-A 21The specific nucleic acid sequence is: 5'-ACTGCTAGAGATTTTCCACATAAAAAAAAAAAAAAAAAA-SH-3'

[0047] (2) Preparation of SA-Bio-DNA T and SA-Bio-DNA C :

[0048] Dissolve streptavidin in pure water, take 20 μL of streptavidin (1 mg / mL) and add it to 20 μL of complementary DNA with a concentration of 100 μM. T and DNA C Then, 10 μL and 0.01 mol / L PBS solution were added respectively, incubated at 4°C for more than 2 h, and then centrifuged at 6000 rpm for 20 min using a 30 kDA ultrafiltration tube. Centrifuge twice in total, invert the inner tube and centrifuge at 1800 rpm for 8 min to collect the liquid and make SA-Bio-DNA T and SA-Bio-DNA C , store at 4℃ for future use.

[0049] The complementary strand DNA T The specific nucleic acid sequence is: 5'-Biotin-ATGTGGAAAATCTCTAGCAGTAAA-3'.

[0050] The complementary strand DNA C The specific nucleic acid sequence is: 5'-Biotin-TTTTTTTTTTTTTTTTTT-3'.

[0051] (3) Preparation of T-line and C-line:

[0052] The prepared SA-Bio-DNA T and SA-Bio-DNA C Use a film sprayer to spray gold on the NC film to make T lines and C lines, and dry them in an oven at 37°C for more than 2 hours;

[0053] (4) Assembling aptamer test strips:

[0054] The sample pad was immersed in the sample pad treatment solution for more than 1 hour, dried at 37°C, and then the sample pad, NC membrane, and absorbent pad were assembled and fixed on a polyethylene base plate, cut into 3.5 mm wide pieces, and sealed and dried for storage; wherein the sample pad treatment solution was a mixed solution containing 0.1% (v / v) Tween-20, 1% (v / v) BSA, and 20 g / L sucrose, and the mixture was diluted with 0.01 mol / L PBS buffer solution.

[0055] (5) Aptamer test strip detection:

[0056] AuNPs@SH-polyA-A21, binding buffer (10mM HEPES, 50mM NaCl, 10mM MgCl2, 10% methanol, pH=6), and sample solution were mixed at a volume ratio of 1:1:8, and the mixture was incubated at 37°C with shaking for 45min. Then, 60μL of the incubation solution was added to the sample pad, and the color development of the T line and C line was observed after the color stabilized.

[0057] Example 2 Establishment of a standard curve for detecting AFM1

[0058] Take 10 μL of the detection probe AuNPs@SH-polyA-A 21 , set the total volume to 100 μL, dilute the AFM1 standard sample with binding buffer (10 mM HEPES, 50 mM NaCl, 10 mM MgCl2, 10% methanol, pH = 6) to a final concentration of 2, 5, 10, 50, 100, 250, 500 ng / mL standard solution, incubate at 37 ° C with shaking for 45 min, take 60 μL and drop it on the test strip prepared in Example 1, wait for 15 min and observe the color development of the T line and C line, take a photo with a portable device (mobile phone), and then use Image J to read the gray value on the T line and C line in the photo, calculate T / C, and perform quantitative analysis of AFM1. The results obtained by the AuNPs labeled aptamer test strip are as follows Figure 4 As shown in the figure, as the concentration of AFM1 increases, the color development of the T line becomes weaker. When the concentration of AFM1 is 500 ng / mL, the T line has almost no color development.

[0059] The grayscale values ​​of the T and C lines of the AFM1 test strips with different concentrations were measured using the Image J software, and T / C was calculated. The detection range of AFM1 was found to be 2-500 ng / mL. The linear equation was then fitted using the Origin software, as shown in Figure 5 According to the linear equation y = -0.36475x + 1.1158, the linear range is 2-500 ng / mL, and the quantitative detection limit of the aptamer lateral flow test strip is 0.26 ng / mL. The above results show that the lateral flow test strip developed by the present invention has a wide detection range, high sensitivity, and low detection limit (LOD), and can achieve stable detection in practical applications.

[0060] Example 3 Specificity detection of test strips

[0061] The AFM1 test strips were specifically tested, and four targets that coexisted with AFM1 in the actual samples were selected (the control detection concentration was 100 ng / mL): ochratoxin (OTA), aflatoxin B1 (AFB1), zearalenone (ZEN), and deoxynivalenol (DON). The total volume of the solution was set to 100 μL, and 10 μL of AuNPs@SH-polyA-A was taken. 21 , prepare a solution with a final concentration of 100 ng / mL, set up a blank control group, incubate at 37°C with shaking for 45 min, take 60 μL and drop it on the test strip, wait for 15 min and observe the color development of the T line and C line. Figure 6 As shown, it can be found that under the same concentration, the color of the T line of the test strip becomes obviously lighter only when AFM1 is present, while the color of the T line is rich when other toxins are present or AFM1 is not present, indicating that the test strip has good specificity for AFM1.

[0062] Example 4 Detection of AFM1 recovery in milk samples

[0063] A type of milk was randomly purchased from a supermarket. After being identified as a positive sample, it was pretreated and prepared into AFM1 standard solutions with final concentrations of 50ng / mL, 100ng / mL, and 250ng / mL, and the pH was adjusted to 6.0. The spike recovery experiment in the actual milk sample was carried out, and the spike recovery rate was 92%-104.35%, and the RSD was 1.4%-2.5%. These results can prove that the test strip can accurately and reliably detect the concentration of AFM1 in milk, and further verify the stability and accuracy of the new paper-based aptamer sensor for detecting AFM1 in practical applications.

[0064] The aptamer lateral flow test strip prepared in Example 1 was used to detect the AFM1 content in milk. The results are shown in Table 1.

[0065] Table 1 Detection of AFM1 in milk samples

[0066] <![CDATA[Final concentration of AFM1 (ng / mL)]]> Test results (ng / mL) RSD(%) Recovery rate (%) 0 ND NC NC 50 49.77 2.5 99.54 100 92.34 1.4 92.34 250 260.84 1.6 104.35

[0067] Note: ND means not detected; NC means not calculated.

[0068] Example 5 Stability and repeatability test of test strips

[0069] The aptamer test strips prepared in Example 1 were stored at room temperature (25°C) in a dry, dark environment for 3 days, 7 days, 14 days, 21 days, 30 days, 2 months, 3 months, 4 months, 5 months, and 6 months to detect positive samples containing AFM1 (repeated three times, with an interval of more than 2 hours between each two times). The T / C obtained from the test strips is highly consistent with that of the new test strips. These results can all prove that the aptamer test strips are batch stable and can maintain their functions at room temperature (25°C) for at least half a year, and the results of the test strips are highly reproducible, as shown in Table 2.

[0070] Table 2 Test strip stability and repeatability test

[0071] Storage time (days) Test results 0 +(3 / 3) 3 +(3 / 3) 7 +(3 / 3) 14 +(3 / 3) 21 +(3 / 3) 30 +(3 / 3) 60 +(3 / 3) 90 +(3 / 3) 120 +(3 / 3) 150 +(3 / 3) 180 +(3 / 3)

[0072] Comparative Example 1:

[0073] The specific implementation method is the same as that of Example 1, except that the streptavidin (1 mg / mL) of Example 1 is also added to the DNA T , DNA C The volume ratio of 1:1 was replaced with 2:1, and the detection effect of the test strip was verified according to the method of Example 4. The results showed that the false positive detection rate was high, and the detection sensitivity, detection range, detection limit and stability were all lower than the test strip prepared in Example 1.

[0074] Comparative Example 2:

[0075] The specific implementation method is the same as Example 1, except that the binding buffer HEPES used in incubating the target and the conjugate in Example 1 is replaced by PBS, Tris, and Tris-HCl, respectively. The detection effect of the test paper is verified according to the method of Example 4, and the performance is compared based on the signal-to-noise ratio (S / N, where S is the relative signal intensity in the absence of AFM1, and N is the relative signal intensity in the presence of AFM1). The results show that when the buffer solution is PBS, Tris, and Tris-HCl, the S / N is 1.25, 1.29, and 0.73, respectively, which is lower than the S / N=1.51 when the binding buffer is HEPES, indicating that the aptamer test strip prepared in Example 1 has higher sensitivity, a wider detection range, and a lower detection limit.

[0076] Comparative Example 3:

[0077] The specific implementation method is the same as Example 1, except that the pH value of the binding buffer solution when the target and the conjugate are incubated in Example 1 is adjusted from 6 to 5, 5.5, 7, 7.5, and 8, respectively. The detection effect of the test paper is verified according to the method of Example 4. According to the S / N comparison performance, the results show that when the buffer solution is 5, 5.5, 7, 7.5, and 8, the S / N is 0.85, 0.53, 1.06, 1.49, and 1.45, respectively, which is lower than the S / N = 1.73 when the pH value of the binding buffer is 6.

[0078] Comparative Example 4:

[0079] The specific implementation method is the same as Example 2, except that the incubation temperature of 37°C in Example 2 is replaced by 4°C and 25°C, and the detection effect of the test paper is verified according to the method of Example 4. The performance is compared based on the signal-to-noise ratio. The results show that when the incubation temperature is 4°C and 25°C, the S / N is 1.10 and 0.66 respectively, which is lower than the S / N=1.78 when the incubation temperature is 37°C.

[0080] Comparative Example 5:

[0081] The specific implementation method is the same as Example 2, except that the incubation time of 45 min in Example 2 is replaced by 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min. The detection effect of the test paper is verified according to the method of Example 4. When the incubation time is 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min, the S / N is 0.599, 0.477, 1.46, 2.32, 1.53, and 0.648, respectively, which is lower than the S / N=2.52 when the incubation time is 45 min. This indicates that the aptamer test strip prepared in Example 1 has a shorter detection time, a higher sensitivity, a wider detection range, and a lower detection limit.

[0082] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing an aflatoxin M1 aptamer test strip based on AuNPs, characterized in that: include: (a) Preparation of AuNPs-aptamer conjugates AuNPs@SH-polyA-A 21 ; (b) Preparation of aptamer complementary strand and streptavidin complex SA-Bio-DNA T and SA-Bio-DNA C : Streptavidin at a concentration of 1 mg / mL was mixed with complementary DNA at a concentration of 100 μM. T and DNA C PBS solution, incubate at 4°C for at least 2 h, and then centrifuge to collect the liquid to make SA-Bio-DNA T and SA-Bio-DNA C ; (c) Preparation of T-line and C-line: The SA-Bio-DNAT and SA-Bio-DNAC prepared in step (b) were sprayed onto the nitrocellulose membrane using a film spraying gold apparatus to form T lines and C lines, respectively, and dried for at least 2 hours; (d) Assembling aptamer test strips: The sample pad was immersed in the sample pad treatment solution for at least 1 hour, and after drying, it was assembled with the NC membrane and the absorbent pad and fixed on the polyethylene base plate.

2. The method according to claim 1, characterized in that: The complementary strand DNA described in step (b) T The specific nucleic acid sequence is: 5'-Biotin-ATGTGGAAAATCTCTAGCAGTAAA-3'; complementary strand DNA C The specific nucleic acid sequence is: 5'-Biotin-TTTTTTTTTTTTTTTTTT-3'.

3. The method according to claim 1 or 2, characterized in that: The sample pad treatment solution described in step (d) is a mixed solution containing Tween-20, BSA and sucrose.

4. The method according to any one of claims 1 to 3, characterized in that: The AuNPs aptamer conjugate AuNPs@SH-polyA-A 21 The SH-polyA-A 21 The nucleic acid sequence is: 5'-ACTGCTAGAGATTTTCCACATAAAAAAAAAAAAAAAAAA-SH-3'.

5. An aflatoxin M1 aptamer test strip based on AuNPs, characterized in that: Containing AuNPs aptamer conjugate AuNPs@SH-polyA-A 21 , an aptamer complementary chain and a streptavidin complex; the aptamer complementary chain and the streptavidin complex include SA-Bio-DNA T and SA-Bio-DNA C ; the SH-polyA-A 21 It has the sequence shown as 5'-ACTGCTAGAGATTTTCCACATAAAAAAAAAAAAAAAAAA-SH-3'.

6. The AuNPs-based aflatoxin M1 aptamer test strip according to claim 5, characterized in that: The invention is prepared by the method according to any one of claims 1 to 4.

7. A method for detecting the content of aflatoxin M1, characterized in that: The sample to be tested was conjugated with the AuNPs aptamer conjugate AuNPs@SH-polyA-A 21 The mixture is mixed in a binding buffer containing HEPES, incubated at 36-38° C. for 45-90 min, and dripped onto the test strip. The aflatoxin M1 content is calculated based on the gray values ​​of the T line and the C line.

8. The method according to claim 7, characterized in that The color development of the test strip was photographed, and the gray values ​​of the T line and the C line were measured using the software Image J. The aflatoxin M1 content was calculated based on the linear relationship between the T / C value and the AFM1 concentration.

9. Use of the AuNPs-based aflatoxin M1 aptamer test strip according to claim 5 or 6, or the method according to claim 7 or 8 in the detection of aflatoxin M1.

10. The use according to claim 7, characterized in that: The application includes but is not limited to the detection of aflatoxin M1 in food.

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