AFB1 detection method based on SERS fusion HCR signal amplification technology

Abstract

The invention belongs to the field of molecular biology, and particularly relates to an AFB1 detection method based on SERS fusion HCR signal amplification technology. According to the invention, gold-coated magnetic nanoparticles (Fe3O4-coated AuNPs) are established as a substrate, and the trace AFB1 is detected on the basis of a strategy of fusing a surface enhanced Raman spectroscopy (SERS) with a hybridization chain reaction (HCR). According to the invention, the specific gene sequence is modified on the gold-coated magnetic nanoparticles to be used as an identification probe of the AFB1, and methylene blue is used as a signal molecule to be embedded between DNA double chains, so that the conversion and amplification efficiency in AFB1 detection is greatly improved; meanwhile, the method does not need to depend on expensive equipment and expensive materials, and has the advantages of high efficiency, environmental protection, simplicity, convenience, high sensitivity and the like; the method has good selectivity and reproducibility, has huge potential in research of AFB1 detection, and has a wide market application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and in particular relates to an AFB1 detection method based on SERS fusion HCR signal amplification technology. Background Art

[0002] As a fundamental food source for humans, the quality and safety of cereals are of paramount importance. According to the national standard GB 2761-2017, the AFB1 concentration in corn and peanuts is 20 μg / kg, and in wheat it is 5 μg / kg. Therefore, developing a rapid, highly sensitive method for detecting AFB1 in cereals that can achieve a low limit of detection is crucial to ensuring human health and safety. Common methods for detecting AFB1 include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS). While these existing methods are widely used due to their high accuracy, versatility, adaptability, and robustness, they also have limitations. These include high cost, the requirement for expensive equipment and skilled technicians, and complex operation. Therefore, there is an urgent need for an efficient, environmentally friendly, simple, and highly sensitive method for trace analysis of AFB1.

[0003] Surface-enhanced Raman scattering (SERS) technology offers advantages such as fast detection speed, narrow spectral bandwidth, and rich fingerprint information. It can identify samples at the molecular level, and its simple sample preparation and operation make it suitable for rapid detection. Therefore, it has been widely used to detect trace amounts of small molecules such as nucleic acids and proteins. However, current detection of AFB1 generally suffers from poor sensitivity and accuracy; therefore, achieving highly sensitive detection of AFB1 using surface-enhanced Raman scattering technology remains a key challenge facing researchers. Summary of the Invention

[0004] In order to solve the above problems and realize trace analysis of AFB1, the present invention provides an AFB1 detection method based on SERS fusion HCR signal amplification technology. The SERS detection results are correlated with HCR-mediated signal amplification, which can significantly improve the signal conversion and amplification efficiency in AFB1 detection, thereby realizing rapid and highly sensitive detection of trace AFB1. This method abandons cumbersome operations, enzyme treatment, interference from complex substances, etc., simplifies the operation process, ensures specificity for the target object, and ensures the accuracy and reliability of the results, providing a powerful tool for the detection of AFB1 in food.

[0005] In order to solve the above technical problems, the present invention is implemented through the following technical solutions:

[0006] A preparation method for an AFB1 detection method based on SERS fusion HCR signal amplification technology comprises the following steps:

[0007] Step 1, preparation of gold nanoparticles: HAuCl4 solution was heated to boiling under magnetic stirring, and then trisodium citrate (C6H5Na3O7) solution was added to react. After the reaction was completed, the solution was cooled to room temperature to obtain Au NPs solution;

[0008] Step 2, synthesis of ferroferric oxide nanoparticles: FeCl3·6H2O, polyethylene glycol 6000, and sodium acetate (NaAc) were mixed with ethylene glycol and stirred to obtain a mixed solution; the mixed solution was transferred to a reactor for reaction, and after the reaction, the solution was cooled to room temperature, and then the solid product was collected by magnetic separation and washed several times with ethanol and ultrapure water, respectively. Finally, the washed product was vacuum dried to obtain a black powder, which is ferroferric oxide nanoparticles, denoted as Fe3O4 NPs;

[0009] Step 3, preparation of Fe3O4@AuNPs: The Fe3O4 NPs obtained in step 2 were added to ultrapure water containing PEI, and the product was collected after ultrasonic and oscillation treatment. The product was then washed with ultrapure water and subjected to magnetic separation to obtain solid particles, which were redispersed in water to obtain a dispersion;

[0010] The dispersion was subjected to magnetic separation and solid particles were collected, which were recorded as Fe3O4@PEI NPs. The Au NPs solution prepared in step 1 was added to the Fe3O4@PEI NPs, and after mechanical shaking (until the solution no longer had a noticeable red color), magnetic separation was performed. The magnetically separated solid particles were washed with Tirs-HCl buffer, and the solid particles obtained after washing were collected and recorded as Fe3O4@AuNPs.

[0011] Step 4: Preparation of capture unit:

[0012] The AFB1 aptamer is abbreviated as AFB1-Apt. AFB1-Apt dry powder is diluted with Tris-HCl buffer to obtain an AFB1-Apt solution. The solid particles Fe3O4@Au NPs obtained in step 3 are then mixed with the AFB1-Apt solution. After freezing, the mixture is shaken at room temperature. After the reaction, the solid particles are collected by magnetic separation and washed with Tris-HCl buffer. The washed solid particles are the capture units that can specifically bind to AFB1 and are denoted as Fe3O4@AuNPs-Apt.

[0013] Step 5: Reaction with target:

[0014] The solid particles Fe3O4@AuNPs-Apt obtained in step 4 were added to the AFB1 standard solution. After incubation, the precipitate was collected by magnetic separation and washed with Tris-HCl buffer. The solid particles were then collected by magnetic separation and recorded as solid particles A.

[0015] Step 6, HCR reaction process and preparation:

[0016] Take two kinds of DNA dry powder, labeled DNA1 and DNA2; then dissolve DNA1 and DNA2 in Tris-HCl buffer respectively to obtain DNA1 solution and DNA2 solution; then anneal to form hairpin structures, labeled H1 and H2 respectively;

[0017] H1 and H2 are mixed with the solid particles A in step 5, and after a secondary reaction, the precipitate is collected by magnetic separation and added to Tris-HCl buffer for washing. Finally, the solid particles are collected by magnetic separation and recorded as solid particles B.

[0018] Step 7, preparation of signal molecules:

[0019] The solid particles B obtained in step 6 are added to a methylene blue solution for incubation. After incubation, the solid particles are collected by magnetic separation, and the solid particles are washed with Tris-HCl buffer and ultrapure water in sequence. After washing, the solid particles are magnetically separated again to collect the solid particles, which are recorded as solid particles C. Finally, the solid particles are dispersed in ultrapure water to obtain solution C.

[0020] Step 8: Testing

[0021] S1: Establishment of standard curve:

[0022] First, the SERS intensity value of solution C in step 7 is measured, and then a standard curve is constructed based on the SERS intensity value and the corresponding AFB1 standard solution concentration;

[0023] S2: Detection of AFB1 content in cereal samples:

[0024] The grains are pretreated to obtain a test solution; then, the same procedures as in steps 5-8 are followed, except that the AFB1 standard solution in step 5 is replaced with the test solution. Finally, the SERS intensity value is measured and applied to the standard curve obtained in step 8 S1 to calculate the AFB1 content in the food.

[0025] Furthermore, in step 1, the concentration of the HAuCl4 solution is 0.01%, the concentration of the C6H5Na3O7 solution is 1%, the volume ratio of the HAuCl4 solution to the C6H5Na3O7 solution is 100:0.75, and the reaction time is 10-60 min.

[0026] Furthermore, in step 2, the dosage of FeCl3·6H2O, polyethylene glycol 6000, NaAc and ethylene glycol is 2.7g:1g:8g:80mL; the stirring time is 1h; the reaction temperature is 200°C, the time is 16h, the washing number is 3-5 times, and the vacuum drying condition is 60°C for 5h.

[0027] Furthermore, in step three, the dosage relationship of Fe3O4 NPs, PEI and ultrapure water is 0.1 g:0.01 g:10 mL, the ultrasonic treatment time is 15-30 min, and the oscillation treatment time is 2 h; the concentration of the dispersion is 1 mg / mL; the volume ratio of the dispersion to the AuNPs solution is 2:5, and the mechanical oscillation time is 5-12 h.

[0028] Furthermore, in step 4, the concentration of the AFB1-Apt solution is 3 μM; the dosage of the AFB1-Apt solution and the Fe3O4@Au NPs is 50 μL:200 mg; the freezing temperature is -20°C, the time is 1-3 hours, and the oscillation reaction time is 8-12 hours;

[0029] The sequence of AFB1-Apt is:

[0030] 5′-SH-TTTTTGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTT CGCTAGGCCC-3′;

[0031] Furthermore, in step 5, the dosage of Fe3O4@AuNPs-Apt and AFB1 standard solution is 200 mg:4 μL, wherein the concentration range of AFB1 standard solution is 0.001-1000 ng / mL; the incubation reaction temperature is room temperature, and the time is 30-60 min.

[0032] Furthermore, the sequences of the nucleotide chains DNA1 and DNA2 in step 6 are:

[0033] H1: 5′-CCCTTCGCTAGGCCCCAAAGTGGGCCTAGCGAAGGGCACGAGA-3′; H2: 5′-ACTTTGGGGCCTAGCGAAGGGTCTCGTGCCCTTCGCTAGGCCC-3′.

[0034] Furthermore, the concentrations of DNA1 solution and DNA2 solution were both 4 μM, the annealing reaction conditions were 95°C, heating for 5 min, cooling to 25°C, and reacting for 2 h to form a hairpin structure; the temperature of the secondary reaction was 30-40°C, the reaction time was 60-120 min, and the dosage relationship of H1, H2 and solid particles A was 50 μL: 50 μL: 200 mg.

[0035] Furthermore, in step seven, the concentration of the methylene blue solution is 20-40 μM, the incubation temperature is room temperature, the time is 20-60 min, and the amount of methylene blue solution, solid particles B and ultrapure water used for dispersion is 100 μL: 200 mg: 10 μL.

[0036] Furthermore, in step 8 S1, the step of detecting the SERS intensity value of the solution is as follows: measuring and recording the SERS intensity value of the C solution at 448 cm under 785 nm excitation light. -1 The SERS intensity value at is the characteristic value of the SERS signal intensity of the detection solution.

[0037] Compared with the existing technology, the beneficial effects of the present invention are as follows:

[0038] 1. The present invention uses Fe3O4@Au NPs modified with AFB1 aptamers as the capture substrate, utilizes the infinite extension of DNA chains for signal amplification, and uses the SERS spectral data of methylene blue embedded in the DNA chain through π bonds as the output. This method can achieve specific recognition of AFB1 in cereals, improving the sensitivity and specificity of detection.

[0039] 2. The AFB1 detection method prepared by the present invention greatly improves the conversion and amplification efficiency in AFB1 detection by correlating SERS spectral data with AFB1 concentration, and successfully realizes rapid and highly sensitive trace analysis of AFB1.

[0040] 3. The AFB1 detection method prepared by the present invention abandons tedious operations, enzyme treatment, interference of complex substances, etc., simplifies the operation process, improves sensitivity, reduces cost and time consumption, ensures the specificity of the experiment, and ensures the accuracy and reliability of the results. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the process of the AFB1 detection method based on SERS fusion HCR signal amplification technology of the present invention.

[0042] Figure 2 (A) is a feasibility study; (B) is a specificity study.

[0043] Figure 3 (A) is the final SERS signal intensity obtained by detecting the concentration of AFB1 from 0.001 to 1000 ng / mL; (B) is a calibration graph of SERS intensity and the logarithm of AFB1 concentration.

[0044] Figure 4 For stability analysis. DETAILED DESCRIPTION

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art in the art. Although the present invention has only described preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0047] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0048] AFB1-Apt and DNA dry powder (including DNA1 and DNA2) were purchased from Sangon Biotech (Shanghai) Co., Ltd.; other materials and reagents, unless otherwise specified, were obtained from commercial sources.

[0049] This embodiment provides an AFB1 detection method based on SERS fusion HCR signal amplification technology, such as Figure 1 The schematic diagram shown intuitively depicts the entire process of applying the AFB1 detection method. Fe3O4@Au NPs modified with the AFB1 aptamer are used as a capture substrate to be incubated with the analyte. After washing, they react with the hairpin structures H1 and H2. After washing, methylene blue is added, and the analyte is dispersed in ultrapure water. After washing, the analyte is detected using a Raman spectrometer to obtain SERS spectral data, thereby realizing the detection of AFB1. The specific operation is detailed in the Examples.

[0050] Example 1:

[0051] The AFB1 detection method based on SERS fusion HCR signal amplification technology includes the following steps:

[0052] Step 1. Synthesis of gold nanoparticles: 100 mL of 0.01% HAuCl4 solution was heated to boiling under magnetic stirring (500 rpm), followed by the addition of 0.75 mL of 1% trisodium citrate solution and the reaction was continued for 30 minutes. After the reaction, the mixture was cooled to room temperature to obtain an AuNPs solution; the diameter of the gold nanoparticles (Au NPs) was approximately 20-30 nm.

[0053] Step 2: Synthesis of Fe3O4 nanoparticles: 2.7 g of FeCl3·6H2O, 1 g of polyethylene glycol 6000, and 8 g of NaAc were mixed with 80 mL of ethylene glycol and stirred vigorously for 1 hour to obtain a mixed solution. The mixed solution was then transferred to a 100 mL reactor and reacted at 200°C for 16 hours. After the reaction, the solution was cooled to room temperature and the solid particles were collected by magnetic separation. The solid particles were washed three times with ethanol and then ultrapure water. Finally, the solid powder was dried in vacuo at 60°C for 5 hours to obtain a black powder, namely Fe3O4 nanoparticles, with a diameter of approximately 150 nm.

[0054] Step 3: Synthesis of gold-coated magnetic nanoparticles: 0.1 g of Fe3O4 NPs obtained in step 2 was added to 10 mL of ultrapure water containing 0.01 g of PEI, and the mixture was ultrasonicated for 15 min and mechanically shaken for 2 h. The solid product was collected and then washed three times with ultrapure water. The solid particles were then magnetically separated and redispersed in ultrapure water to obtain a dispersion with a concentration of 1 mg / mL.

[0055] Take 200 μL of the dispersion for magnetic separation and collect the solid particles, which are recorded as Fe3O4@PEI NPs; then add 500 μL of the AuNPs solution prepared in step 1 to the Fe3O4@PEI NPs; mechanically shake overnight (10 h), collect the solid particles by magnetic separation, and then wash them three times with Tris-HCl buffer. Finally, collect the obtained solid particles by magnetic separation again, which are Fe3O4@AuNPs, and store them at 4°C for use.

[0056] Step 4. Synthesis of AFB1-Apt-modified gold-coated magnetic nanoparticles: The AFB1 aptamer is abbreviated as AFB1-Apt. AFB1-Apt dry powder was diluted with Tris-HCl buffer to obtain a 3 μM concentration of AFB1-Apt. Then, 50 μL of the AFB1-Apt solution was mixed with 200 mg of Fe3O4@Au NPs and frozen at -20°C for 2 hours to obtain the resulting mixture. After freezing, the mixture was shaken at room temperature overnight. The solid particles were then collected by magnetic separation and washed three times with Tris-HCl buffer. Magnetic separation was then repeated to obtain AFB1-Apt-modified gold-coated magnetic nanoparticles, abbreviated as Fe3O4@Au NPs-Apt.

[0057] The nucleotide sequence of AFB1-Apt is:

[0058] 5′-SH-TTTTTGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCT AGGCCC-3′,

[0059] Step 5: Reaction with target:

[0060] 200 mg of the solid particles Fe3O4@Au NPs-Apt obtained in step 4 were incubated with 4 μL of AFB1 standard solution (0.001-1000 ng / mL) at room temperature for 30 min. The precipitate was collected by magnetic separation and then washed three times with Tris-HCl buffer. Finally, magnetic separation was performed again to collect the solid particles, which were recorded as solid particles A.

[0061] Step 6: Prepare the hairpin DNA (H1, H2) and methylene blue solution required for the HCR reaction: H1 and H2 are the hairpin DNAs used for HCR, and methylene blue is a signal molecule that is embedded in the DNA double strand through a π bond;

[0062] (1) The specific synthesis steps of hairpin DNA (H1, H2) are as follows: take two kinds of DNA dry powder, marked as DNA1 and DNA2; dilute the two DNA dry powders to 4 μM with Tris-HCl buffer to obtain DNA1 solution and DNA2 solution respectively; then use a PCR instrument to perform annealing reaction, that is, heating at 95°C for 5 minutes, and then cooling to 25°C for 2 hours to form hairpin structures, which are marked as H1 and H2 respectively;

[0063] The sequences of nucleotide chains DNA1 and DNA2 are:

[0064] H1: 5′-CCCTTCGCTAGGCCCCAAAGTGGGCCTAGCGAAGGGCACGAGA-3′; H2: 5′-ACTTTGGGGCCTAGCGAAGGGTCTCGTGCCCTTCGCTAGGCCC-3′.

[0065] (2) The steps for the hybridization chain reaction (HCR) are as follows: 50 μL of H1 and 50 μL of H2 were added to 200 mg of solid particles A, respectively, and reacted at 30°C for 90 min to form long double-stranded DNA. The solid particles were collected by magnetic separation, and then washed three times with Tris-HCl to remove unreacted H1 and H2. Finally, the solid particles were collected by magnetic separation and recorded as solid particles B.

[0066] Step 7: Preparation of methylene blue as a signal molecule:

[0067] Weigh methylene blue and dissolve it in ultrapure water to prepare a 30 mM methylene blue solution;

[0068] 100 μL of 30 mM methylene blue solution was added to 200 mg of solid particles B obtained in step 6, and the mixture was incubated at room temperature for 20 min. After incubation, the solid particles were collected by magnetic separation, and then washed with Tris-HCl buffer until the solution was colorless, and then washed with ultrapure water until the solution was colorless. After washing, the solution was magnetically separated again to obtain solid particles, which were recorded as solid particles C. The obtained solid particles C were redispersed in 10 μL of ultrapure water and recorded as solution C.

[0069] The nucleotide sequences of the hairpin structures DNA1 and DNA2 are:

[0070] DNA1:

[0071] 5′-CCCTTCGCTAGGCCCCAAAGTGGGCCTAGCGAAGGGCACGAGA-3′,

[0072] DNA2:

[0073] 5′-ACTTTGGGGCCTAGCGAAGGGTCTCGTGCCCTTCGCTAGGCCC-3′

[0074] Control group (Without AFB1): Follow steps 5-7, except that the AFB1 standard solution in step 5 is replaced with an equal volume of deionized water. The resulting solution is designated as solution C0 and serves as the control group.

[0075] 1. Feasibility Study:

[0076] Figure 2 (A) is a feasibility study, divided into experimental group, namely C solution (WithAFB1); control group, namely C0 solution (WithoutAFB1); Results Figure 2 As shown in Figure (A), compared with the SERS intensity without the target AFB1, the SERS intensity is significantly reduced after the target is added, indicating that the present invention can be used for the detection of AFB1;

[0077] 2. Specificity detection:

[0078] To further demonstrate that the detection method designed by the present invention can specifically detect AFB1, the following experiment was conducted: aflatoxin B2 (AFB2), ochratoxin (OTA), zearalenone (ZEN), deoxynivalenol (DON), and fumonisin B1 (FAB1) were selected as controls; a mixed group (MAX) was also established, where MAX refers to the same concentration of AFB2, OTA, ZEN, DON, FAB1 mixed with AFB1 for detection. The test results are as follows: Figure 2As shown in Figure (B), the results show that no interference can cause a significant change in the SERS intensity, indicating that the SERS-based detection method has excellent specificity for AFB1.

[0079] Step 8: SERS spectrum detection:

[0080] S1: Establishment of standard curve:

[0081] The characteristic peak of solution C was detected at 448 cm -1 The Raman intensity at , and the standard curve between the SERS intensity value and the corresponding AFB1 standard solution concentration was established; Figure 3 As shown in Figure A, the Raman signal intensity decreases with the increase of AFB1 concentration, and there is a linear correlation between the Raman signal intensity and AFB1 concentration ( Figure 3 B). The regression equation is y=-769.4946x+5068.8832(R 2 =0.9976), where y is the Raman signal intensity and x is the logarithm of the AFB1 concentration. The detection limit is as low as 0.907 pg / mL, indicating that this method can still effectively detect AFB1 at extremely low concentrations and is suitable for trace analysis of AFB1.

[0082] S2: Detection of AFB1 content in cereal samples:

[0083] To further demonstrate that the detection method designed by the present invention can effectively detect AFB1 in cereals, the specific steps are as follows:

[0084] (1) The pretreatment steps for grain samples (specifically wheat, corn, and peanuts) are as follows: 5.0 g of grain sample was dissolved in a 50 mL centrifuge tube; the sample was shaken vigorously with 20 mL of methanol / distilled water (70:30 v / v) and 0.5 g of NaCl for 30 min, then centrifuged, the supernatant was collected and filtered through a 0.22 μM membrane. Finally, the residual extract was redissolved in distilled water / methanol (80:20, v / v) to obtain the test solution; and the analysis was performed using this method, that is, the operations of steps 5 to 8 were followed, except that the AFB1 standard solution in step 5 was replaced with the test solution. The SERS intensity value was finally measured and applied to the standard curve obtained in S1 of step 8 to calculate the concentration of AFB1.

[0085] (2) The results of the detection method designed by the present invention were compared with those of the spiked grain samples tested using the national standard method. The results are as follows: HPLC-MS and the detection results of this study were analyzed for three foods: corn, peanuts, and wheat. The AFB1 concentrations used were 0.10, 1.00, and 10.0 ng / mL, respectively. The recovery rates of wheat ranged from 87.24% to 114.05%, with RSDs of 7.36% to 9.91%; the recovery rates of corn ranged from 90.92% to 95.60%, with RSDs of 6.06% to 13.58%; and the recovery rates of peanuts ranged from 101.05% to 117.98%, with RSDs of 9.91% to 13.93%. This demonstrates the feasibility and accuracy of the detection method designed by the present invention in practical applications.

[0086] Table 1: Detection recovery

[0087]

[0088] a Mean, the average value of the detection.

[0089] b SD, standard deviation.

[0090] To further demonstrate the stability and reproducibility of the method designed in this invention for AFB1 detection, the reproducibility of the method was evaluated by performing three replicate measurements of the target substance, AFB1. The relative standard deviations (RSDs) of the three replicate measurements were 6.8, 9.2, and 2.1%, respectively, demonstrating the good reproducibility of the method for AFB1 detection.

[0091] In addition, Fe3O4@AuNPs-Apt prepared in step 4 and H1 and H2 prepared in step 6 were stored at 4°C for 6 groups. One group was used to prepare solution C every week for 6 consecutive weeks, and the SERS intensity of solution C was recorded every week. The test results are shown in Figure 2. Figure 4 As shown, the SERS signal intensity did not change significantly, indicating that the AFB1 detection method based on the SERS fusion HCR signal amplification technology of the present invention has excellent stability.

[0092] In summary, this method innovatively combines hybridization chain reaction (HCR) for signal amplification, including rapid magnetic separation, HCR-based signal amplification for trace analysis, and SERS signal output using methylene blue. It exhibits good specificity, stability, and reproducibility. The results indicate that the SERS-infused HCR signal amplification-based detection method has great potential for AFB1 detection, outperforming traditional AFB1 detection methods in terms of efficiency, sensitivity, specificity, and reliability. Therefore, this easily prepared AFB1 detection method with signal amplification has broad potential for application in food testing.

[0093] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although this specification has described the present invention in detail with reference to the above embodiments, it should be understood by those skilled in the art that the present invention may still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for detecting AFB1 based on SERS fusion HCR signal amplification technology, characterized in that: Here are the steps: Step 1: Heat the HAuCl4 solution to boiling under magnetic stirring, then add trisodium citrate solution to react, and after the reaction is completed, cool the solution to room temperature to obtain an AuNPs solution; Step 2: FeCl3·6H2O, polyethylene glycol 6000, and sodium acetate are mixed with ethylene glycol and stirred to obtain a mixed solution; the mixed solution is transferred to a reactor for reaction, and the solution is cooled to room temperature after the reaction. The solid product is then collected by magnetic separation and washed several times with ethanol and ultrapure water, respectively. Finally, the washed product is vacuum dried to obtain a black powder, namely, ferrosoferric oxide nanoparticles, denoted as Fe3O4NPs. Step 3: Add the Fe3O4 NPs obtained in step 2 to ultrapure water containing PEI, collect the product after ultrasonic and oscillation treatment, wash it with ultrapure water, and then perform magnetic separation to obtain solid particles, which are redispersed in water to obtain a dispersion; The dispersion was subjected to magnetic separation and solid particles were collected, which were recorded as Fe3O4@PEI NPs. The Au NPs solution prepared in step 1 was added to the Fe3O4@PEI NPs, and magnetic separation was performed after mechanical shaking. The magnetically separated solid particles were washed with Tirs-HCl buffer, and the solid particles obtained after washing were collected and recorded as Fe3O4@Au NPs. Step 4: The AFB1 aptamer is abbreviated as AFB1-Apt. The AFB1-Apt dry powder is diluted with Tris-HCl buffer to obtain an AFB1-Apt solution. The solid particles Fe3O4@Au NPs obtained in step 3 are then mixed with the AFB1-Apt solution. After freezing, the mixture is shaken at room temperature. After the reaction, the solid particles are collected by magnetic separation and washed with Tris-HCl buffer. The washed solid particles are the capture units that can specifically bind to AFB1 and are denoted as Fe3O4@Au NPs-Apt. Step 5: The solid particles Fe3O4@Au NPs-Apt obtained in step 4 were added to the AFB1 standard solution. After incubation, the precipitate was collected by magnetic separation and washed with Tris-HCl buffer. The solid particles were then collected by magnetic separation and recorded as solid particles A. Step 6: Take two kinds of DNA dry powder, labeled DNA1 and DNA2; then dissolve DNA1 and DNA2 in Tris-HCl buffer respectively to obtain DNA1 solution and DNA2 solution; then anneal them to form hairpin structures, labeled H1 and H2 respectively; Mix H1 and H2 with the solid particles A in step 5, perform a secondary reaction, collect the precipitate by magnetic separation, add Tris-HCl buffer for washing, and finally collect the solid particles by magnetic separation, which are recorded as solid particles B; Step 7: The solid particles B obtained in step 6 are added to a methylene blue solution for incubation. After incubation, the solid particles are collected by magnetic separation, and the solid particles are washed with Tris-HCl buffer and ultrapure water in sequence. After washing, the solid particles are magnetically separated again to collect the solid particles, which are recorded as solid particles C. Finally, the solid particles are dispersed in ultrapure water to obtain solution C. Step 8: Testing S1: Establishment of standard curve: First, the SERS intensity value of solution C in step 7 is measured, and a standard curve is established between the SERS intensity value and the corresponding AFB1 standard solution concentration; S2: Detection of AFB1 content in cereal samples: The grains are pretreated to obtain the test solution; then the operations of steps 5-8 are followed, except that the AFB1 standard solution in step 5 is replaced with the test solution. Finally, the SERS intensity value is measured and applied to the standard curve obtained in step S1 to calculate the AFB1 content in the food.

2. The AFB1 detection method based on SERS fusion HCR signal amplification technology according to claim 1, characterized in that: In step 1, the concentration of the HAuCl4 solution is 0.01%, the concentration of the trisodium citrate solution is 1%, the volume ratio of the HAuCl4 solution to the C6H5Na3O7 solution is 100:0.75, and the reaction time is 10-60 minutes.

3. The AFB1 detection method based on SERS fusion HCR signal amplification technology according to claim 1, characterized in that: In step 2, the dosage of FeCl3·6H2O, polyethylene glycol 6000, sodium acetate and ethylene glycol is 2.7g:1g:8g:80mL; the stirring time is 1h; the reaction temperature is 200°C, the reaction time is 16h, the washing number is 3-5 times, and the vacuum drying condition is 60°C for 5h.

4. The AFB1 detection method based on SERS fusion HCR signal amplification technology according to claim 1, characterized in that: In step 3, the dosage of Fe3O4 NPs, PEI and ultrapure water is 0.1 g:0.01 g:10 mL, the ultrasonic treatment time is 15-30 min, and the oscillation time is 2 h; the concentration of the dispersion is 1 mg / mL; the volume ratio of the dispersion to the Au NPs solution is 2:5, and the mechanical oscillation time is 5-12 h.

5. The AFB1 detection method based on SERS fusion HCR signal amplification technology according to claim 1, characterized in that: In step 4, the concentration of the AFB1-Apt solution is 3 μM; the dosage of the AFB1-Apt solution and Fe3O4@AuNPs is 50 μL:200 mg; the freezing temperature is -20°C, the time is 1-3 hours, and the oscillation reaction time is 8-12 hours; The sequence of AFB1-Apt is: 5′-SH-TTTTTGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTT CGCTAGGCCC-3′.

6. The AFB1 detection method based on SERS fusion HCR signal amplification technology according to claim 1, characterized in that: In step 5, the dosage of Fe3O4@AuNPs-Apt and AFB1 standard solution is 200 mg:4 μL, wherein the concentration range of AFB1 standard solution is 0.001-1000 ng / mL; the incubation reaction temperature is room temperature, and the time is 30-60 min.

7. The AFB1 detection method based on SERS fusion HCR signal amplification technology according to claim 1, characterized in that: The sequences of the nucleotide chains DNA1 and DNA2 in step 6 are: H1:5′-CCCTTCGCTAGGCCCCAAAGTGGGCCTAGCGAAGGGCACGAGA-3′; H2: 5′-ACTTTGGGGCCTAGCGAAGGGTCTCGTGCCCTTCGCTAGGCCC-3′.

8. The AFB1 detection method based on SERS fusion HCR signal amplification technology according to claim 1, characterized in that: In step six, the concentrations of DNA1 solution and DNA2 solution are both 4 μM, the annealing reaction conditions are 95°C, heating for 5 minutes, cooling to 25°C, and reacting for 2 hours to form a hairpin structure; the temperature of the secondary reaction is 30-40°C, the reaction time is 60-120 minutes, and the dosage relationship of H1, H2 and solid particles A is 50 μL: 50 μL: 200 mg.

9. The AFB1 detection method based on SERS fusion HCR signal amplification technology according to claim 1, characterized in that: In step seven, the concentration of the methylene blue solution is 20-40 μM, the incubation temperature is room temperature, the time is 20-60 min, and the amount of methylene blue solution, solid particles B and ultrapure water used for dispersion is 100 μL: 200 mg: 10 μL.

10. The AFB1 detection method based on SERS fusion HCR signal amplification technology according to claim 1, characterized in that: In step S1, the steps of detecting the SERS intensity value of the solution are as follows: measuring and recording the SERS intensity value of the C solution at 448 cm under the excitation of 785 nm excitation light. -1 The SERS intensity value at is the characteristic value of the SERS signal intensity of the detection solution.

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