A fluorescence aptamer sensing analysis method for aflatoxin B1 based on single-walled carbon nanohorns

Abstract

The present invention belongs to the field of food safety biological detection, and in particular, to a fluorescence aptamer sensing analysis method for aflatoxin B1 based on single-walled carbon nanohorns. Aiming at the problems that the existing high-performance liquid chromatography method relies on large-scale instruments, the immunoassay operation is cumbersome and not suitable for on-site rapid detection, and the aptamer-based biosensor method uses aptamers as specific recognition elements, the following scheme is proposed, which includes the following steps: S1. When AFB1 is present, the aptamer modified with a fluorescent group specifically recognizes and binds to form a stable secondary structure. The present invention mainly consists of an AFB1 aptamer modified with a fluorescent group as a fluorescent probe and SWCNHs as a quencher. Utilizing the unique property of SWCNHs to adsorb ssDNA nucleic acid aptamers promotes the fluorescence release when AFB1 is present and the fluorescence quenching when AFB1 is absent. Optimize various detection parameters to realize the detection of real samples contaminated with AFB1.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety biological detection, and particularly relates to a fluorescence aptamer sensing analysis method for aflatoxin B1 based on single-walled carbon nanohorns. Background Art

[0002] Aflatoxin is a kind of mycotoxin, with carcinogenic, teratogenic, mutagenic, nephrotoxic and hepatotoxic effects. Aflatoxins contaminated under natural conditions mainly include four types: B1, B2, G1 and G2, among which B1 is the most, followed by G1, and B2 and G2 are very few. Because aflatoxin B1 (AFB1) has the greatest toxicity and carcinogenicity, AFB1 is generally used as the main index when evaluating the content of aflatoxin in feed and grain. Due to the negative impact of mycotoxins on human and animal health, it has received extensive attention in the past 10 to 15 years. Therefore, many countries have formulated regulations and standards to strictly control the maximum residue limits of mycotoxins in food and feed. At the same time, the International Agency for Research on Cancer classifies AFB1 as a Class I carcinogen, and OTA and FB1 are classified as Class IIB carcinogens. Thus, it can be seen that doing a good job in the inspection and detection of food safety is of great significance for ensuring human food safety. Conventional methods for AFB1 mainly include chromatography and immunoassay, such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). Although these conventional methods have the advantages of high selectivity and high sensitivity, their disadvantages cannot be ignored. For example, chromatography requires precise instruments, complex pretreatment and professional operators, and the relative insufficiency of antibodies in immunoassay limits the wide application of these traditional methods. Therefore, there is an urgent need to develop a simple, economical and relatively fast analytical method to replace them.

[0003] Some new detection techniques, such as biosensor method, have the advantages of high detection sensitivity, fast analysis speed, low cost and simple operation, and have been widely used in the detection of aflatoxin in food. Among them, a biosensor is a sensor device that uses the specific recognition process of a biorecognition element and combines the signal transduction process of a physical or chemical conversion element to achieve detection. In the construction of biosensors, nucleic acid aptamers are often used as biorecognition elements due to their unique advantages. An aptamer is a synthetic oligonucleotide (ssDNA or ssRNA) sequence of about 30-80 bases in length screened from an in vitro random DNA or RNA library by the systematic evolution of ligands by exponential enrichment (SELEX) technology. It can specifically bind to the target under certain conditions and has the advantages of a wide target range, strong specificity, strong stability, high affinity, easy modification, etc. compared with antibodies. Therefore, it is widely used as a recognition element in the field of analytical detection. Therefore, aptamer-based biosensors can overcome the limitations of the above chromatographic and immunoassay methods and provide a more convenient detection strategy for AFB1 detection. In recent years, nanomaterials have been widely used in aptamer sensors due to their unique physical properties and chemical stability. Single-walled carbon nanohorns (SWCNHs) are a new type of carbonaceous nanomaterial, which is a conical structure starting from a pentacyclic structure and expanding outward with a hexagonal graphite structure. Compared with ordinary nanomaterials, it has a larger specific surface area and better dispersibility, which increases the application of SWCNHs. It has been confirmed by research that the nucleic acid base rings on ssDNA can bind to the surface structure of SWCNHs through π-π covalent bond stacking, so that ssDNA is adsorbed on the surface of SWCNHs. However, due to the hydrogen bond interaction between the nucleic acid bases of the phosphate backbone in dsDNA, dsDNA can no longer interact with SWCNHs. Based on this characteristic, SWCNHs have great research value in the application of aptamer sensors.

[0004] In the past two years, great progress has been made in the field of aptamer sensing technology for AFB1 toxin detection. Various new sensing strategies have emerged continuously and demonstrated excellent sensitivity and specificity. These studies have brought infinite possibilities to the aptamer field. However, some challenges in actual detection limit its wide application. Among them, the problems of weak anti-interference ability, high cost and low detection sensitivity are relatively prominent. In addition, in real toxin contamination events, the actual samples usually involve complex matrices, which increases the difficulty of detection.

[0005] In this study, a novel fluorescence aptamer sensing method will be constructed by combining SWCNHs for the rapid detection of AFB1. This detection method mainly consists of an AFB1 aptamer modified with FAM (FAM-Apt) as a fluorescence probe and SWCNHs as a quencher. The sensor is designed by utilizing the unique property of SWCNHs to adsorb ssDNA aptamers. When AFB1 is present, FAM-Apt preferentially binds specifically with AFB1 to form a stable complex, and the failure of SWCNHs to adsorb the FAM-Apt and AFB1 complex results in the presence of fluorescence. When AFB1 is absent, FAM-AFB1 will be adsorbed by SWCNHs, leading to fluorescence quenching. In addition, the sensor is simple in structure, high in stability, easy to operate, and low in detection cost, and is very suitable for non-professional people to rapidly detect AFB1 contamination in food on-site. Summary of the Invention

[0006] The object of the present invention is to solve the disadvantages in the prior art that high-performance liquid chromatography relies on large instruments, and the immunoassay is cumbersome to operate and not suitable for on-site rapid detection, and to propose a fluorescence aptamer sensing analysis method for aflatoxin B1 based on single-walled carbon nanohorns.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A fluorescence aptamer sensing analysis method for aflatoxin B1 based on single-walled carbon nanohorns, comprising the following steps:

[0009] S1. When AFB1 is present, the aptamer modified with a fluorescent group specifically binds to it to form a stable secondary structure;

[0010] S2. The secondary structure can resist the adsorption of SWCNHs, so that the aptamer and the single-walled carbon nanohorns SWCNHs cannot approach each other, and thus the fluorescence resonance energy transfer effect cannot be triggered and the fluorescence signal cannot be quenched. At this time, the fluorescence signal in the system is strong;

[0011] S3. When AFB1 is absent, the aptamer is in a free single-stranded DNA state and is adsorbed on the surface of SWCNHs, so that the single-stranded aptamer modified with a fluorescent group and SWCNHs trigger the fluorescence resonance energy transfer effect and the fluorescence signal is quenched;

[0012] S4. By detecting the change in the fluorescence signal, the quantitative detection of AFB1 is realized.

[0013] Preferably, in S1, for the treatment of AFB1: Since the AFB1 aptamer adheres to the tube wall in the form of a very light dry film, centrifugation is required before opening to prevent loss. The specific operation is as follows: Centrifuge at 4000 rpm for 30 s - 60 s, then slowly open the tube cap, add an appropriate amount of buffer solution according to experimental needs, cover the cap and shake well, and dispense a small amount into several centrifuge tubes for storage at 20 °C. Take it as needed and avoid repeated freezing and thawing.

[0014] Preferably, during the subsequent experiment, the AFB1 aptamer needs to be heat-treated to a certain extent. Heat the aptamer solution dissolved to room temperature at 90 °C for 10 min, then ice-bath for 10 min, and let it stand at room temperature for 10 min before the experiment. Set 3 parallel experiments for each group and conduct the experiment under light-shielded conditions to protect the stability of the FAM group.

[0015] Preferably, in S2, optimize the concentration of SWCNHs, incubation time, and incubation temperature. Since SWCNHs has an adsorption effect on single-stranded nucleic acid aptamers and can quench fluorescence, the nucleic acid aptamer labeled with a fluorescent group will bind to SWCNHs and fluorescence quenching will occur in the absence of the target. Conversely, when the nucleic acid aptamer binds to AFB1, the fluorescence is restored.

[0016] Preferably, the optimization of the SWCNHs concentration is as follows: Add SWCNHs with different mass ratios to the pretreated AFB1 aptamer. The mass ratios of SWCNHs to AFB1 aptamer are 0:1, 50:1, 100:1, 200:1, 300:1, 400:1, 600:1 respectively. Oscillate and incubate the mixture of AFB1 aptamer and SWCNHs, centrifuge at 12000 rpm for 15 min, and then measure the fluorescence value of the supernatant. Among them, the group without adding SWCNHs is used as the control group, and the fluorescence quenching rate (F0 - F) / F0 is used as the analysis index, where F is the experimental group and F0 is the control group.

[0017] Preferably, the optimization of the incubation time is as follows: Optimize two incubation times; one is the incubation time of AFB1 aptamer and SWCNHs. Add an appropriate amount of SWCNHs to the pretreated AFB1 aptamer, oscillate and incubate the mixture of AFB1 aptamer and SWCNHs for 5, 10, 20, 30, 40, 50 min, centrifuge at 12000 rpm for 15 min, and then measure the fluorescence value of the supernatant. Among them, the group without adding SWCNHs is used as the control group, F is the experimental group, and F0 is the control group.

[0018] Preferably, second, the incubation time of the AFB1 aptamer and AFB1. Add AFB1 to the pretreated AFB1 aptamer, incubate at an appropriate temperature for 30, 60, 90, 120, 150 min, then add SWCNHs, perform oscillatory incubation for 30 min, centrifuge at 12,000 rpm for 15 min, and measure the fluorescence value of the supernatant. Among them, use the binding buffer to replace the target as the blank control group, take the relative fluorescence difference as the ordinate and time as the abscissa for analysis.

[0019] Preferably, the optimization of the incubation temperature is as follows: Mix the pretreated aptamer solution and AFB1 in a centrifuge tube, incubate at 4, 20, 30, 37, 45 °C for 2 h respectively, then add an appropriate amount of SWCNHs solution, incubate for 30 min, centrifuge, and measure the fluorescence value of the supernatant. Among them, use the binding buffer to replace the target as the blank control group, take the relative fluorescence difference as the ordinate and time as the abscissa for analysis.

[0020] Preferably, in S1, for the quantitative analysis and detection of AFB1, the specific operation is as follows: Add AFB1 standard products with different concentrations of 0 - 500 ng / mL to the pretreated aptamer solution, perform oscillatory incubation at 37 °C for 2 h, then add an appropriate amount of SWCNHs solution, perform oscillatory incubation at 37 °C for 30 min, and finally make up to 500 μL with the binding buffer solution. Among them, use the binding buffer to replace the target as the blank control group. After the incubation is completed, the mixture is centrifuged at 12,000 rpm for 15 min, take the supernatant, measure the fluorescence intensity with an enzyme-labeled instrument at an excitation wavelength of 495 nm and an emission wavelength of 520 nm, take the target concentration as the abscissa and the relative fluorescence difference as the ordinate to draw a standard curve.

[0021] Preferably, when SWCNHs binds to the AFB1 aptamer, fluorescence resonance energy transfer occurs, resulting in fluorescence quenching, and it shows different adsorption effects on DNA strands in different structural states.

[0022] In the present invention, the beneficial effects of the fluorescence aptamer sensing analysis method for aflatoxin B1 based on single-walled carbon nanohorns are as follows:

[0023] The present invention combines SWCNHs to construct a novel fluorescence aptamer sensing detection method for rapid detection of AFB1, which mainly consists of a fluorescence group-modified AFB1 aptamer as a fluorescence probe and SWCNHs as a quencher. Utilize the unique property of SWCNHs to adsorb ssDNA nucleic acid aptamers to promote the phenomenon of fluorescence release when AFB1 is present and fluorescence quenching when AFB1 is absent, optimize various detection parameters, and realize the detection of real samples contaminated with AFB1. Description of the Drawings

[0024] Figure 1 Schematic diagram of the AFB1 detection by the SWCNHs-based fluorescence sensor of the present invention;

[0025] Figure 2 Optimization diagram of the mass ratio of SWCNHs to aptamer of the present invention;

[0026] Figure 3 Optimization diagram of the incubation time of AFB1 aptamer and SWCNHs of the present invention;

[0027] Figure 4 Optimization diagram of the incubation time of AFB1 aptamer and AFB1 of the present invention;

[0028] Figure 5 Optimization diagram of the incubation temperature of the present invention;

[0029] Figure 6 Standard curve diagram of AFB1 of the present invention;

[0030] Figure 7 Specificity analysis diagram of AFB1 aptamer of the present invention. Detailed implementation manners

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0032] Referring to Figure 1-7 , a fluorescence aptamer sensing and analysis method for aflatoxin B1 based on single-walled carbon nanohorns, the specific steps are as follows:

[0033] Single-walled carbon nanohorns SWCNHs can bind to the AFB1 aptamer to cause fluorescence resonance energy transfer and result in fluorescence quenching, showing different adsorption effects on DNA strands in different structural states. When AFB1 is present, the aptamer modified with a fluorescent group (FAM) specifically recognizes and binds to it to form a stable secondary structure, which can resist the adsorption of SWCNHs, so that the aptamer and SWCNHs cannot approach each other, and thus cannot trigger the fluorescence resonance energy transfer effect and quench the fluorescence signal. At this time, the fluorescence signal in the system is strong. When AFB1 is absent, the aptamer is in a free single-stranded DNA state and is adsorbed on the surface of SWCNHs, causing the single-stranded aptamer modified with a fluorescent group to trigger the fluorescence resonance energy transfer effect with SWCNHs, and the fluorescence signal is quenched. Therefore, by detecting the change of the fluorescence signal, the quantitative detection of AFB1 can be realized.

[0034] AFB1 aptamer pretreatment:

[0035] Configuration of AFB1 aptamer solution: Since the AFB1 aptamer (5’-FAM-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3’) adheres to the tube wall in the form of a very light dry film, centrifugation is required before opening to prevent loss. The specific operation is as follows: Centrifuge at 4000 rpm for 30 s - 60 s, then slowly open the tube cap, add an appropriate amount of buffer solution according to the experimental needs, cover the cap and shake well, aliquot a small amount into several centrifuge tubes and store at 20 °C. Take it as needed, avoid repeated freezing and thawing. When conducting subsequent experiments, the AFB1 aptamer needs to be heat-treated to a certain extent. Heat the aptamer solution dissolved to room temperature at 90 °C for 10 min, then ice-bath for 10 min, and let it stand at room temperature for 10 min before the experiment. Each group of experiments is set up with 3 parallel experiments and carried out under light-shielded conditions to protect the stability of the FAM group.

[0036] To obtain the best experimental results, it is necessary to optimize the system conditions in the constructed detection method, including relevant experimental conditions such as the concentration of SWCNHs, incubation time, incubation temperature, etc. Since SWCNHs has an adsorption effect on single-stranded nucleic acid aptamers and can quench fluorescence at the same time, the nucleic acid aptamer labeled with a fluorescent group will bind to SWCNHs and fluorescence quenching will occur in the absence of the target. On the contrary, when the nucleic acid aptamer binds to AFB1, the fluorescence is restored.

[0037] Optimization of SWCNHs concentration:

[0038] To determine the optimal concentration of SWCNHs required for the reaction system, different mass ratios of SWCNHs are added to the pretreated AFB1 aptamer. The mass ratios of SWCNHs to AFB1 aptamer are 0:1, 50:1, 100:1, 200:1, 300:1, 400:1, 600:1 respectively. The mixture of AFB1 aptamer and SWCNHs is incubated with shaking. After centrifuging at 12000 rpm for 15 min, the fluorescence value of the supernatant is measured, with the group without adding SWCNHs as the control group. The fluorescence quenching rate (F0-F) / F0 is used as the analysis index (F is the experimental group, F0 is the control group).

[0039] Figure 2 Shows the fluorescence quenching rate between different mass ratios of SWCNHs and AFB1 aptamer. As the mass ratio increases, the fluorescence value in the supernatant gradually decreases, and the fluorescence quenching rate continuously increases. After the mass ratio of SWCNHs to the aptamer increases to 200:1, the growth rate of the fluorescence quenching rate suddenly drops, indicating that the fluorescence of the AFB1 aptamer is basically completely quenched by SWCNHs. Therefore, in order to ensure that the AFB1 aptamer is completely adsorbed by SWCNHs, 300:1 (m SWCNHs : mssDNA ) as the optimal mass ratio.

[0040] Optimization of the incubation time

[0041] To determine the effect of the incubation time on the fluorescence intensity in the reaction system, this experiment optimized two incubation times:

[0042] (I) Incubation time of AFB1 aptamer and SWCNHs. An appropriate amount of SWCNHs was added to the pretreated AFB1 aptamer, and the mixture of AFB1 aptamer and SWCNHs was incubated with shaking for 5, 10, 20, 30, 40, 50 min. After centrifugation at 12000 rpm for 15 min, the fluorescence value of the supernatant was measured, with the group without adding SWCNHs as the control group (F is the experimental group, F0 is the control group).

[0043] (II) Incubation time of AFB1 aptamer and AFB1. AFB1 was added to the pretreated AFB1 aptamer and incubated at an appropriate temperature for 30, 60, 90, 120, 150 min, then SWCNHs was added and incubated with shaking for 30 min. After centrifugation at 12000 rpm for 15 min, the fluorescence value of the supernatant was measured. The binding buffer was used to replace the target as the blank control group, and the relative fluorescence difference was used as the ordinate and time as the abscissa for analysis.

[0044] In addition, during the optimization of the incubation time, Figure 3 it was shown that the fluorescence quenching rate reached the highest value of 93% after the AFB1 aptamer and SWCNHs were incubated for 30 min, and there was no greater fluctuation in the fluorescence recovery rate within a longer time interval. Therefore, 30 min was selected as the optimal quenching time for the AFB1 aptamer. In addition, the optimal reaction time between the AFB1 aptamer and AFB1 was also determined. As Figure 4 shown, as the incubation time gradually increased, the fluorescence value of the supernatant continuously increased, and the relative fluorescence intensity also increased accordingly. When the incubation time increased to 120 min, the relative fluorescence intensity reached the maximum. Therefore, 120 min was selected as the optimal reaction time between the AFB1 aptamer and AFB1.

[0045] Optimization of the incubation temperature:

[0046] To determine the optimal temperature in the constructed sensing system, the pretreated aptamer solution and AFB1 were mixed evenly in a centrifuge tube and incubated at 4, 20, 30, 37, 45 °C for 2 h respectively. Then an appropriate amount of SWCNHs solution was added and incubated for 30 min. After centrifugation, the fluorescence value of the supernatant was measured. The binding buffer was used to replace the target as the blank control group, and the relative fluorescence difference was used as the ordinate and time as the abscissa for analysis.

[0047] The experimental results are as Figure 5The results show that the relative fluorescence intensity is lowest at 4°C and reaches the maximum at 37°C, indicating that the binding effect of nucleic acid aptamer and AFB1 is best at this temperature. Therefore, in the detection system established in this experiment, 37°C was selected as the optimal temperature of the entire reaction system.

[0048] In order to further verify the sensitivity of the constructed sensing method, it is necessary to measure various performances of the sensor, such as detection limit, specificity, and actual sample measurement.

[0049] AFB1 standard curve drawing:

[0050] AFB1 was quantitatively analyzed and detected under the optimized optimal system conditions. The specific operation was as follows: different concentrations of AFB1 standard (0-500ng / mL.) were added to the pretreated aptamer solution and incubated at 37°C for 2h. Then, an appropriate amount of SWCNHs solution was added and incubated at 37°C for 30min. Finally, the binding buffer solution was added to 500μL, in which the binding buffer was used to replace the target as a blank control group. After the incubation, the mixture was centrifuged at 12000rpm for 15min, and the supernatant was taken. The fluorescence intensity was measured by an ELISA reader at an excitation wavelength of 495nm and an emission wavelength of 520nm. The standard curve was drawn with the target concentration as the horizontal axis and the relative fluorescence difference as the vertical axis.

[0051] like Figure 6 As shown in the figure, with the increase of AFB1 concentration, the fluorescence intensity also gradually increased, and the relative fluorescence increase was linear in the range of 10-100 ng / mL AFB1 concentration. Linear regression analysis was used, and the fitting equation was Y = 7.1104X [AFB1] +98.39754, R 2 =0.99202. Based on the calculation of three times the standard deviation, the LOD of this detection method is 4.1 ng / mL.

[0052] Aptamer specificity assay:

[0053] To verify the selectivity of the established method, specificity analysis was performed. Common fungal toxins in food that are harmful to human health, such as ochratoxin A (OTA), zearalenone (ZEA), T-2 toxin, and fumonisin (FB1), were selected for selective analysis. To test interference, a specific concentration (50 ng / mL) of each interfering fungal toxin was mixed with the aptamer (where the concentration of AFB1 was 20 ng / mL) using the same optimized conditions as for AFB1 detection. The binding buffer was used instead of the target as a blank group, and the fluorescence of the relative fluorescence difference was analyzed.

[0054] Depend on Figure 7It can be seen that when the concentration of AFB1 is relatively low, the relative fluorescence difference is significantly higher than that of other experimental groups without AFB1. The experimental results show that in the presence of AFB1, the aptamer can bind to the stable secondary structure and is not adsorbed by SWCNHs, thus enhancing the fluorescence value. Therefore, the aptamer sensing detection system established in this experiment has good specificity for AFB1.

[0055] Detection experiment of AFB1 in actual samples:

[0056] To verify the application feasibility of the SWCNHs aptamer sensor in actual samples, a spike recovery experiment was carried out on soybean oil. First, the soybean oil was pretreated. Weigh 5 g of soybean oil and add 25 mL of methanol extraction solution (V 甲醇 :V 水 = 60:40). Mix evenly at room temperature for 25 min, centrifuge at 8500 rpm for 5 min, take the supernatant and filter it twice with a microfiltration membrane, and add 40 mL of binding buffer to obtain the pretreated soybean oil solution. Different concentrations of spiked samples (10 ng / mL, 50 ng / mL, 100 ng / mL) were prepared by adding different concentrations of AFB1 standard solution to the pretreated soybean oil solution, and fluorescence detection was carried out according to the constructed sensing method. At the same time, a control experiment was carried out with an ELISA kit for AFB1 to verify the accuracy of the constructed sensor.

[0057] As shown in Table 1, the recovery rate of the soybean oil samples was 94.1%-109.2%, and the relative standard deviation (RSD) ranged from 4.26% to 5.58%. Comparing the experimental results with the ELISA detection results, the difference in recovery rates was not significant, and in some cases, the RSD was even smaller, indicating that the constructed fluorescence sensing method is accurate and reliable and can sensitively and accurately detect AFB1 toxin in real samples.

[0058] Table 1 Determination of aflatoxin B1 in soybean oil by fluorescence method and ELISA method

[0059]

[0060] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and all should be covered within the protection scope of the present invention.

Claims

1. A fluorescence aptamer sensing and analysis method for aflatoxin B1 based on single-walled carbon nanohorns, characterized in that, It includes the following steps: S1. When AFB1 exists, the aptamer modified with a fluorescent group specifically recognizes and binds to it to form a stable secondary structure; S2. The secondary structure can resist the adsorption of SWCNHs, so that the aptamer and single-walled carbon nanohorns (SWCNHs) cannot approach each other, and thus cannot trigger the fluorescence resonance energy transfer effect and quench the fluorescence signal. At this time, the fluorescence signal in the system is strong; S3. When AFB1 does not exist, the aptamer is in a free single-stranded DNA state and is adsorbed on the surface of SWCNHs, causing the single-stranded aptamer modified with a fluorescent group and SWCNHs to trigger the fluorescence resonance energy transfer effect and quench the fluorescence signal; S4. By detecting the change in the fluorescence signal, the quantitative detection of AFB1 is realized.

2. The fluorescence aptamer sensing and analysis method for aflatoxin B1 based on single-walled carbon nanohorns according to claim 1, wherein In S1, the treatment of AFB1: Since the AFB1 aptamer adheres to the tube wall in a very light dry film state, it needs to be centrifuged before opening to prevent loss. The specific operation is as follows: Centrifuge at 4000 rpm for 30 s - 60 s, then slowly open the tube cap, add 216 μL of TE buffer to each OD of primer to prepare a 10 μM stock solution, cover the cap and shake well, aliquot a small amount into several centrifuge tubes and store at 20 °C, and take it as needed to avoid repeated freezing and thawing.

3. A fluorescence aptamer sensing and analysis method for aflatoxin B1 based on single-walled carbon nanohorns according to claim 2, characterized in that When conducting subsequent experiments, the AFB1 aptamer needs to be heat-treated to a certain extent. Heat the aptamer solution dissolved to room temperature at 90 °C for 10 min, then ice-bath for 10 min, and let it stand at room temperature for 10 min before the experiment. Each group of experiments is set up with 3 parallel experiments and carried out under dark conditions to protect the stability of the FAM group.

4. A fluorescence aptamer sensing and analysis method for aflatoxin B1 based on single-walled carbon nanohorns according to claim 3, characterized in that, In S2, the concentration of SWCNHs, incubation time, and incubation temperature are optimized. Since SWCNHs have an adsorption effect on single-stranded nucleic acid aptamers and can quench fluorescence at the same time, the nucleic acid aptamer labeled with a fluorescent group will bind to SWCNHs and cause fluorescence quenching in the absence of the target. Conversely, when the nucleic acid aptamer binds to AFB1, the fluorescence is restored.

5. A fluorescence aptamer sensing and analysis method for aflatoxin B1 based on single-walled carbon nanohorns according to claim 4, characterized in that The optimization of the SWCNHs concentration is specifically as follows: Add SWCNHs with different mass ratios to the pretreated AFB1 aptamer. The mass ratios of SWCNHs to AFB1 aptamer are 0:1, 50:1, 100:1, 200:1, 300:1, 400:1, 600:1 respectively. Oscillate and incubate the mixture of AFB1 aptamer and SWCNHs, centrifuge at 12000 rpm for 15 min, and then measure the fluorescence value of the supernatant. Among them, the group without adding SWCNHs is used as the control group, and the fluorescence quenching rate (F0 - F) / F0 is used as the analysis index, where F is the experimental group and F0 is the control group.

6. A fluorescence aptamer sensing and analysis method for aflatoxin B1 based on single-walled carbon nanohorns according to claim 5, characterized in that, The optimization of the incubation time is as follows: Two incubation times were optimized. First, the incubation time of AFB1 aptamer and SWCNHs. An appropriate amount of SWCNHs was added to the pretreated AFB1 aptamer, and the mixture of AFB1 aptamer and SWCNHs was incubated with shaking for 5, 10, 20, 30, 40, 50 min. After centrifugation at 12000 rpm for 15 min, the fluorescence value of the supernatant was measured. Among them, without adding SWCNHs was used as the control group, F was the experimental group, and F0 was the control group.

7. A fluorescence aptamer sensing and analysis method for aflatoxin B1 based on single-walled carbon nanohorns according to claim 6, characterized in that, Second, the incubation time of AFB1 aptamer and AFB1. AFB1 was added to the pretreated AFB1 aptamer and incubated at an appropriate temperature for 30, 60, 90, 120, 150 min. Then, SWCNHs were added and incubated with shaking for 30 min. After centrifugation at 12000 rpm for 15 min, the fluorescence value of the supernatant was measured. Among them, using binding buffer to replace the target was used as the blank control group, and the relative fluorescence difference was used as the ordinate and time as the abscissa for analysis.

8. A fluorescence aptamer sensing and analysis method for aflatoxin B1 based on single-walled carbon nanohorns according to claim 7, characterized in that, The optimization of the incubation temperature is as follows: The pretreated aptamer solution and AFB1 were mixed evenly in a centrifuge tube and incubated at 4, 20, 30, 37, 45 °C for 2 h respectively. Then, an appropriate amount of SWCNHs solution was added and incubated for 30 min. After centrifugation, the fluorescence value of the supernatant was measured. Among them, using binding buffer to replace the target was used as the blank control group, and the relative fluorescence difference was used as the ordinate and time as the abscissa for analysis.

9. A fluorescence aptamer sensing and analysis method for aflatoxin B1 based on single-walled carbon nanohorns according to claim 8, characterized in that, In S1, quantitative analysis and detection of AFB1 were carried out. The specific operation is as follows: Different concentrations of AFB1 standard products from 0 - 500 ng / mL were added to the pretreated aptamer solution and incubated with shaking at 37 °C for 2 h. Then, an appropriate amount of SWCNHs solution was added and incubated with shaking at 37 °C for 30 min. Finally, it was made up to 500 μL with binding buffer solution. Among them, using binding buffer to replace the target was used as the blank control group. After the incubation was completed, the mixture was centrifuged at 12000 rpm for 15 min, and then the supernatant was taken. The fluorescence intensity was measured by an enzyme-labeled instrument at an excitation wavelength of 495 nm and an emission wavelength of 520 nm. The target concentration was used as the abscissa and the relative fluorescence difference was used as the ordinate to draw a standard curve.

10. A fluorescence aptamer sensing and analysis method for aflatoxin B1 based on single-walled carbon nanohorns according to claim 9, characterized in that, The binding of SWCNHs and AFB1 aptamer causes fluorescence resonance energy transfer, resulting in fluorescence quenching, and shows different adsorption effects on DNA strands in different structural states.

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