General nucleic acid aptamer for broad-spectrum recognition of aflatoxin, screening optimization method and application

Abstract

The invention belongs to the technical field of molecular biology and poison detection, and provides a universal nucleic acid aptamer for broad-spectrum recognition of aflatoxin and a screening optimization method and application of the universal nucleic acid aptamer. The nucleic acid aptamer is a single-stranded DNA molecular sequence shown in SEQ ID NO.3. The nucleic acid aptamer specifically recognizes aflatoxin, can be combined with AFB1, AFB2, AFM1 and AFM2, and can be used for detecting aflatoxin. The method has the universality of simultaneously identifying aflatoxin substances. A good linear relation with AFB1 in the range of 0.031 ng / mL to 46.84 ng / mL is shown, and LOD is 0.0060 ng / mL. A good linear relation with AFM1 in a range of 0.033 ng / mL to 32.83 ng / mL is shown, and LOD is 0.010 ng / mL. The developed HCR / CuNPs-MoS2Ns sensing system can be used for detecting the AFB1 and the AFM1 in an actual sample.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and poison detection technology, and specifically relates to a universal nucleic acid aptamer for broad-spectrum recognition of aflatoxin and a screening and optimization method and application thereof. The nucleic acid aptamer is obtained based on molecular dynamics screening and optimization, and specifically recognizes aflatoxin, and has the versatility of simultaneous recognition of aflatoxin-like substances. Background Art

[0002] Aflatoxins (AFT) are common mycotoxins, secondary metabolites secreted by different types of filamentous fungi, which can cause acute or chronic poisoning in humans and animals, damage the liver, kidneys, nerve tissue, hematopoietic tissue and skin tissue. Aflatoxins are a type of anti-nutritional factor that is widely present in various agricultural products, food, feed and Chinese medicinal materials, seriously contaminating peanuts, corn, soybeans, wheat and other grain and oil products. They are the most toxic of many mycotoxins and are extremely harmful to humans. More than 20 types of AFT have been discovered, and the most common aflatoxins are B 1 , B 2 , G 1 , G 2 and M 1 、M 2 There are six types of aflatoxin, of which aflatoxin B is the most toxic and harmful 1 (Aflatoxin B 1 , AFB1, AFM1), over the years, AFB1, AFM1 poisoning caused deaths of people and animals in various countries around the world, which seriously threatens human health and life safety and affects the regional economy and import and export trade of many countries. The detection methods of aflatoxin mainly include instrumental analysis, immunoassay and other analysis. With the progress and development of science and technology, different detection technologies have made great progress in repeatability, stability, sensitivity and other aspects. Although instrumental analysis methods such as high performance liquid chromatography and mass spectrometry have high accuracy, sensitivity and good repeatability, they are expensive and have complex sample pretreatment, which is not suitable for large-scale sample analysis; methods based on immunoassay such as immunoassay strips and test strips can realize on-site rapid detection of large quantities of samples, but the test results are prone to discrepancies, with high probability of false positives and false negatives, which can easily affect the stability and reliability of the results; TLC method is simple to operate among other analysis methods, but can only achieve qualitative semi-quantitative detection. In actual poison detection work, it is still necessary to develop faster and more sensitive detection technologies, and it is urgent to establish a method suitable for sensitive detection of toxic markers in samples. As a technology with great potential, biosensor technology has become the focus of current research on rapid detection of aflatoxin.

[0003] Aptamers are single-stranded DNA or RNA with specific base sequences obtained through artificial screening by the exponential enrichment (SELEX) system, which can specifically bind to a variety of targets. Compared with traditional antibodies, aptamers not only have higher biological affinity for the target, but also have the advantages of low acquisition cost, high stability, easy modification, and strong immunogenicity. They are used as recognition elements of a variety of biosensors. Under suitable conditions, aptamers bind to the target, isomerize from a free coiled state to a unique secondary or tertiary structure, and form a stable target-aptamer complex. The binding of the target to the aptamer usually acts on a specific region, which may be a part of the aptamer, thereby confirming the presence of redundant nucleotides in the aptamer. The original aptamer with a long oligonucleotide sequence may bind to its target analog through the primer region or additional fragments, resulting in reduced specificity. Therefore, by optimizing the truncation of the original oligonucleotide sequence, developing efficient aptamers with higher affinity, specificity and low cost, while reducing the synthesis cost, it is also beneficial to improve the sensitivity of the biosensor system. In recent years, there have been reports on the screening or truncation of aflatoxin nucleic acid aptamers for the construction of biosensors with different mechanisms for the detection of aflatoxin and its derivatives. The nucleic acid aptamer sequences used in these related studies are relatively long, which limits their use.

[0004] In the single-stranded nucleic acid aptamer technology, high sensitivity of DNA detection can be achieved by synthesizing double-stranded DNA and adding DNA amplification technology, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and rolling circle amplification (RCA), but these technologies require the use of corresponding enzymes, and they are very susceptible to the environment and toxic chemicals. Summary of the invention

[0005] The present invention provides a universal nucleic acid aptamer for broad-spectrum recognition of aflatoxin, a screening and optimization method and an application thereof. The nucleic acid aptamer is obtained based on molecular dynamics screening and optimization, specifically recognizes aflatoxin, and has the versatility of simultaneous recognition of aflatoxin-like substances.

[0006] The present invention is achieved by the following technical scheme: a universal nucleic acid aptamer for broad-spectrum recognition of aflatoxin, wherein the nucleic acid aptamer is a single-stranded DNA molecular sequence as shown in SEQ ID NO.3 in the nucleotide sequence table, and the specific sequence is GAACGTATTATCACTATGAGTCTCGTTC.

[0007] Based on molecular dynamics screening and optimization of the method for the universal nucleic acid aptamer for broad-spectrum recognition of aflatoxins as described in claim 1, the secondary structure of the 50nt aflatoxin B1 aptamer AF50 is simulated using the online software Mfold web server, which consists of a 20-base ring structure, a 10-base neck structure, and 20 bases at both ends, and its nucleotide sequence is shown in SEQ ID NO.1; the stem-loop structure is retained and the bases away from the two ends are deleted to obtain a truncated 28nt aptamer AF28, and its nucleotide sequence is shown in SEQ ID NO.2; Then, RNA Composer software was used to obtain the tertiary structure of AF28. At the same time, combined with AutoDock 4.2.2 software, the binding pockets of AF28 with aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) were determined through molecular simulation docking program. By mutating some bases of AF28 in the stem and repeating the above molecular docking steps, the structurally stable optimized aptamer T-AF28 was obtained, and its nucleotide sequence is shown in SEQ ID NO.3. Finally, LigPlot+ and PyMOL 2.3.3 software were used to visualize the molecular docking results and determine the core recognition regions of T-AF28 / AFB1 and AF28 / AFM1.

[0008] Furthermore, the stem-loop structure was retained and the bases far from both ends were deleted to obtain the truncated 28nt aptamer AF28. The specific method was as follows: 5 bases at the 5′ end and 17 bases at the 3′ end of the stem-loop region far from AF50 were removed to obtain the aptamer AF28.

[0009] The present invention also provides the use of the universal nucleic acid aptamer for broad-spectrum recognition of aflatoxin in the preparation of a product for detecting aflatoxin.

[0010] Furthermore, the universal nucleic acid aptamer that can broadly recognize aflatoxin is used in the preparation of reagents or drugs for detecting aflatoxin.

[0011] Furthermore, the universal nucleic acid aptamer that can broadly recognize aflatoxins is used in the preparation of drugs or reagents for qualitative and / or quantitative detection of AFB1 and AFM1.

[0012] Furthermore, the universal nucleic acid aptamer for broad-spectrum recognition of aflatoxins is used in the preparation of a kit for qualitative and / or quantitative detection of AFB1 and AFM1.

[0013] Furthermore, the universal nucleic acid aptamer that can broadly recognize aflatoxins is used in the preparation of a biosensor for detecting AFB1 and AFM1.

[0014] Furthermore, the biosensor is a biosensor for detecting AFB1 and AFM1 developed by fluorescence method based on molybdenum disulfide nanosheet signal amplification technology; the biosensor is composed of T-AF28 and its partially complementary oligonucleotide chain cDNA, hairpin 1, namely HP1, hairpin 2, namely HP2, and MoS 2 Ns and in situ prepared CuNPs are used as fluorescent probes; the nucleotide sequence of the cDNA is shown in the sequence table SEQ ID NO.4; the nucleotide sequence of the hairpin probe H1 is shown in the sequence table SEQ ID NO.5, and the nucleotide sequence of the hairpin probe H2 is shown in the sequence table SEQ ID NO.6.

[0015] The biosensor is HCR / CuNPs-MoS 2 The Ns aptamer sensing system is as follows: 0.5 μM T-AF28 and its complementary chain cDNA were dissolved in MOPS buffer at pH 7.4, heated at 37°C for 60 min, and then cooled to room temperature; different concentrations of AFB1 or AFM1 were added, incubated at 37°C for 60 min, and then 0.20-0.50 μM hairpin probes H1 and H2 were introduced, incubated at 37°C for 120 min, and then 0.50 mM copper ions and 0.75 mM ascorbic acid were added, and incubated at room temperature for 10 min; finally, 50 μg / mL MoS 2 Ns was added to the sensing system, and the fluorescence spectrum was measured after standing for 5 min.

[0016] The nucleic acid aptamer of the present invention is simulated by molecular dynamics, and the three-dimensional structure of T-AF28 / aflatoxin analogs shows that T-AF28 can bind to aflatoxin analogs (AFB1, AFB2, AFM1 and AFM2), and T-AF28 has a strong affinity for aflatoxin analogs. In addition, from the predicted secondary structure of T-AF28, it can be seen that the C4, G5 and T23 bases are all located near the stem loop, and the binding pocket of T-AF28 is located in the stem loop region, which plays an important role in the structural stability of the T-AF28 / aflatoxin analog complex.

[0017] The fluorescence intensity of the sensor system showed a good linear relationship with the AFB1 concentration in the range of 0.031 ng / mL to 46.84 ng / mL (y = 126.59x+362.73, R 2 = 0.992, and the detection limit (LOD) was calculated to be 0.0060 ng / mL. In addition, the sensor has the same detection performance for AFM1. The fluorescence of the sensing system also increases with the increase of AFM1 concentration, and has a good linear relationship with the AFM1 concentration in the range of 0.033 ng / mL to 32.83 ng / mL (y = 124.21x+361.15, R2 =0.993), and the detection limit (LOD) was calculated to be 0.010 ng / mL. Compared with the reported detection results of AFB1 and AFM1, the constructed aptamer sensor has satisfactory detection performance, which may be due to the specific interaction between T-AF28 and AFB1 / AFM1 and the interaction between HCR and MoS 2 Ns signal amplification. T-AF28 may be a universal nucleic acid aptamer that can simultaneously identify aflatoxin-like substances. Other toxins such as DON, ZEN and OTA only caused a small fluorescence response in the sensing system. Further studies showed that the developed sensing system had good specificity for aflatoxin-like substances in the presence of other toxins. In addition, for actual samples, other possible coexisting amino acids and ions were explored, including Cys, Tyr, Gly, Trp, Ile, Glu, Ser, Phe, Val, Asp, Gln, Pro, Asn, His, Lys, Thr, Na + Mg 2+ , K + , Ca 2+ , Fe 2+ , Fe 3+ , Cu 2+ 、Zn 2+ , Mn 2+ 、Al 3+ Cr 3+ 、Cd 3+ , Hg 2+ and Ag + , to confirm the selectivity and specificity of the sensing system. Fig.13 B and Fig.13 As shown in C, the fluorescence response of the detection system to AFB1 and AFM1 was significantly higher than that of other potential co-existing interferences, indicating that the sensor system is specific to AFB1 and AFM1. In addition, the AFB1 and AFM1 mixture with co-interference of the control showed similar fluorescence response to AFB1 and AFM1, further indicating that the proposed method is specific and selective to AFB1 and AFM1.

[0018] At the same time, compared with the detection results based on commercial ELISA kits, there was no significant difference in the detection performance of the two methods (the error was less than 10.0%), indicating that the developed HCR / CuNPs-MoS 2 The Ns sensing system can be used to detect AFB1 and AFM1 in actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Middle: A is the molecular docking simulation of AF50 and its truncation mutation scheme; B is the three-dimensional structure of AF50, AF28 and T-AF28 binding to various targets based on molecular docking simulation; Figure 2 Middle: A is the ITC titration of aptamer AF50 binding to AFB1; B is the ITC titration of AF28 binding to AFB1; C is the ITC titration of T-AF28 binding to AFB1; D is the ITC titration of aptamer T-AF28 and AFM1; E is the thermodynamic information of the interaction between T-AF28 and AFB1 and AFM1 determined by ITC titration; Figure 3 Circular dichroism spectra of T-AF28 (0.50 µM) in the absence of AFB1 (10 μM, red line) and AFM1 (10 μM, indigo line); Figure 4 MD simulation diagrams of AF50, T-AF28, T-AF28 / AFB1, and T-AF28&AFM1 complexes; in the figure: A is the RMSD curve; B is the RMSF curve; C is the rotation radius curve; D is the energy decomposition of the important bases of T-AF28 and AFB1 calculated by the MM / PBSA method; E is the energy decomposition of the important bases of T-AF28 and AFM1 calculated by the MM / PBSA method; F is the total interaction energy of T-AF28 with AFB1 and AFM1; Figure 5 Molecular dynamics simulations for (left) three-dimensional structure and (right) predicted secondary structure of the binding pocket formed by (A) AFB1 (B) AFB2 (C) AFM1 (D) AFM2 and T-AF28 (inset of the red box: hydrogen bonding (yellow dashed line), π-π stacking (indigo dashed line) and hydrophobic (gray dashed line) interactions between T-AF28 and aflatoxin analogs. The binding site of T-AF28 and aflatoxin analogs is marked in blue in the secondary structure); Figure 6 HCR-CuNPs / MoS 2 Sensing strategies of AFB1 and AFM1 in Ns aptamer sensing system; Figure 7 The effects of buffer solution type (A) and pH (B) on the sensing system; Figure 8 is the effect of MoS2 concentration on the sensing system; Fig. 9 The effects of T-AF28 / cDNA (A) and H1 / H2 (B) concentrations on AFB1 detection; Fig.10 Optimization of AFB1 / T-AF28 incubation time (A) AFB1 / T-AF28 (B) cDNA / H1 / H2; Fig.11 Optimization of (A) copper ion and (B) ascorbic acid concentrations in the sensing system; Fig.12 Scatter plot of the fluorescence response of the constructed HCR-CuNPs / MoS2Ns aptamer sensing system with the concentration of (A) AFB1 and (C) AFM1; the fluorescence spectrum of the sensing system changes with the increase of AFB1 or AFM1 concentration; (B, D) the fluorescence intensity of the sensing system changes with the concentration of AFB1 (B) and AFM1 (D) and the linear fitting curve; Fig.13 HCR-CuNPs / MoS 2 Selectivity and specificity of the Ns aptamer sensing system; In the figure: A is the specificity of the sensing system to AFB1 and its analogs AFB2, AFM1 and AFM2; B is the specificity of the sensing system to amino acids; C is the specificity of the sensing system to other ions; The concentrations of AFB1, AFB2, AFM1, and AFM2 are all 0.50 µM, and the concentrations of other toxins, amino acids, and ions are all 5.0 µM. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0022] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0023] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0024] 1. Screening and optimization of broad-spectrum nucleic acid aptamers that can identify aflatoxin B1 poisoning markers based on molecular dynamics simulation: A. Experimental Methods 1. Truncated nucleic acid aptamers based on molecular docking: The truncation and mutation of nucleic acid aptamers were performed based on molecular docking technology. The specific steps are as follows: The secondary structure of the aflatoxin B1 aptamer AF50 (50 nt) was simulated using the online software Mfold web server. Then, the truncated aptamer AF28 (28nt) was obtained by retaining the stem-loop structure and deleting the bases away from both ends. The tertiary structure of AF28 was then obtained using RNA Composer software. At the same time, the binding pockets of AF28 with aflatoxin B1, i.e. AFB1, and aflatoxin M1, i.e. AFM1, were determined by molecular simulation docking program in combination with AutoDock 4.2.2 software. By mutating some bases of AF28 in the stem and repeating the above molecular docking steps, the optimized aptamer T-AF28 with stable structure was obtained. Finally, the molecular docking results were visualized and analyzed using LigPlot+ and PyMOL 2.3.3 software, and the core recognition regions of T-AF28 / AFB1 and AF28 / AFM1 were determined.

[0025] 2. Determination of binding affinity based on isothermal titration calorimetry: The titration calorimetry experiment was determined using an iTC200 (MalvernPanalytical, USA) instrument. The solutions used were vented before the experiment. AFB1 or AFM1 reference solution was placed in the titration needle, and T-AF28 buffer solution was placed in the sample pool. The concentration ratio of the reference solution to the aptamer solution was 20:1. To avoid the influence of dilution heat during the titration process, the buffer used in the titration process was consistent. Finally, the titration calorimetry analysis software was used to obtain the enthalpy change (ΔH), entropy change (ΔS) and dissociation constant (Kd) values ​​of AFB1 and AFM1 in the binding of the aptamer T-AF28.

[0026] 3. Circular dichroism: Circular dichroism is an ideal method for studying conformational changes of nucleic acids. The circular dichroism of this experiment uses a BioLogic MOS-500 spectrometer (French), with a spectral bandwidth of 2 nm, a scanning rate of 1 s / point, a scanning range of 200-400 nm, and a 1 cm quartz cuvette for detection at room temperature. T-AF28 (0.5 μM) was dissolved in Tris-HCl (pH=7.4), and then 10 μM AFB1 and 10 μM AFM1 solutions were added to the solution to make a volume of 3 mL, and the mixed solution was incubated at room temperature for 10 min before detection.

[0027] 4. Molecular dynamics: The accuracy of the molecular docking results was further verified by molecular dynamics (MD) simulations, and the interactions between the adapter T-AF28 and aflatoxin B1 and M1 were explored. Due to the structural tension of the ligand-target complex and the rough placement of the water solvent model, the MD system needs to be fully optimized. First, the spatial structure of the optimized aptamer T-AF28 was obtained according to the literature. Then, the ligand structure was energy optimized to eliminate steric hindrance. After minimization, heating and equilibration of the T-AF28-AFB1 and T-AF28-AFM1 complexes, the trajectories within 20ns of the generation phase were calculated and analyzed, and the trajectories were analyzed using root mean square fluctuations and gyration radius.

[0028] 5. Gel electrophoresis: DNA strands were heated to 95°C for 10 min and cooled to room temperature for at least 60 min before use. 1% agarose gel was prepared with 1 TBE buffer (40 mM Tris AcOH, 2.0 mM Na2EDTA, pH 8.5), and M5Hipure NextIII Gelred 10000x was mixed with the sample as a DNA stain. The gel was run at room temperature at 50 V for 90 min. 10 μL of sample was loaded into each layer and photographed on Gel Imaging (Tanon 2500 R, Tianneng Co., Ltd., Shanghai, China).

[0029] B. Experimental Results 1. AFB1 aptamer truncation and mutation: In order to obtain an effectively truncated AFB1-aptamer, it is first necessary to identify the key binding domains of the aptamer and AFB1. Regions with specific secondary structures, such as stem-loop structures, G-quadruplexes, protrusions and / or pseudoknots, are usually the binding domains of the target molecule. Figure 1 As shown in Figure A, the simulation docking model of the 50-base AFB1 aptamer (AF50) was simulated using Mfold. The model consists of a 20-base ring structure, a 10-base neck structure, and 20 bases at both ends. Figure 1 As shown in the purple box in B, the docking model shows that the binding region between AF50 and AFB1 is located on the loop close to the stem region in the AF50 three-dimensional (3D) structure, and G10 is involved in the binding interaction.

[0030] On this basis, guided by the binding information, the long sequence AF50 (50 nt) of the AFB1 aptamer was truncated by retaining the loop structure and part of the stem structure and deleting the redundant bases away from the two ends to obtain the truncated aptamer AF28 (28nt). Then, some base pairs were further mutated in the stem region to obtain a structurally stable optimized aptamer that can recognize both AFB1 and AFM1, which was labeled T-AF28 (28nt).

[0031] The truncation principle is as follows Figure 1 As shown in A. First, the 5 bases at the 5′ end and the 17 bases at the 3′ end of the stem-loop region far from AF50 were removed to obtain the aptamer AF28. According to the titration experiment, the Kd value of the aptamer AF28 for AFB1 was calculated to be 8.85 μM. Compared with the 14.53 μM of the aptamer AF50, the affinity of the aptamer AF28 for AFB1 increased by 1.64 times, which is due to the reduction of the redundant base pairs at both ends ( Figure 1 ). Subsequently, the simulated aptamer AF28 was docked with AFB1 again. According to the binding domain information, the three bases near the 3′ end (purple oval box) were mutated to obtain the aptamer T-AF28 (Kd=0.98 μM), and the affinity was increased by nearly 15 times compared with AF50 ( Figure 2 The affinity of the mutant T-AF28 aptamer was 9-fold higher than that of the truncated AF28, which may be due to the formation of stable complementary chains at both ends of the base pairs.

[0032] In addition, molecular docking revealed that T-AF28 can also bind to AFM1, AFB2, and AFM2, with Kd values ​​of 0.68 uM, 1.01 uM, and 0.86 uM, respectively, and the binding region is consistent with AFB1, which may be related to their similar molecular structures ( Figure 1 The specific thermodynamics of the binding process between aptamers (T-AF28, AF50 and AF28) and AFB1 were studied by isothermal titration calorimetry experiments. The results are shown in Figure 2 and Table 1, where the ΔH and -TΔS of AF50 binding to AFB1 are -9.01 kJ / mol and 727.5 kJ / mol, respectively. It is known that ΔH represents the contribution of specific interactions, and -TΔS represents the contribution of non-specific conformational transitions or steric effects. Compared with AF50, the -TΔS of AF28 binding to AFB1 (349.6 kJ / mol) is smaller than that of AF50 (727.5 kJ / mol), indicating that removing the redundant bases at both ends of AF50 can reduce the interference of conformational changes or spatial movement during binding to AFB1. After some bases in the stem region of AF28 mutated, the affinity of the aptamer T-AF28 to AFB1 was further enhanced, and the binding values ​​-TΔS of T-AF28 to AFB1 (35.7 kJ / mol) and AFM1 (5.29 kJ / mol) were smaller than those of AF28 (349.6 kJ / mol), which may be due to the role of the bases in the stem in stabilizing the recognition structure during the binding process.

[0033] Table 1 ITC titration thermodynamic information of aptamer-target binding In addition, it can be seen from Table 1 that the structural stability of T-AF28 after binding to AFB1 and AFM1 is the highest (△G = -11.87 kJ / mol and -75.72 kJ / mol), which may be due to the base complementary pair formed at the neck position making the complex structure more stable. In summary, the redundant bases at both ends of AF50 were systematically truncated and removed, and the three bases near the 3' end of the truncated base were mutated to obtain the AFB1 and AFM1 aptamer T-AF28 (28 nt) with good affinity. The nucleic acid aptamer sequences used are shown in Table 2.

[0034] Table 2. Aptamer sequence list 2. Circular dichroism: Circular dichroism (CD) is a powerful tool to study the interaction between aptamers and their targets. Figure 3 As shown in the figure, T-AF28 has positive and negative peaks of the cotton effect at 240 nm and 280 nm, respectively, indicating that T-AF28 belongs to a typical b-type DNA. It should be noted that the negative peak of the cotton effect is generated by the helical structure of DNA, while the positive peak of the cotton effect is generated by the stacking of DNA bases. When combined with AFB1 or AFM1, the peak intensities of the cotton effect of T-AF28 at 240 nm and 280 nm are enhanced, indicating that a more compact b-type DNA is formed. The presence of AFB1 or AFM1 stabilizes the structure of T-AF28, which is consistent with literature reports.

[0035] 3. Molecular dynamics simulation: Molecular dynamics (MD) simulation is often used to study conformational changes over time in biological systems. The root mean square deviation (RMSD) is often used as a tool to measure the degree of conformational difference or ballistic stability. Figure 4 A depicts the RMSD curve of T-AF28 in the absence of AFB1 and AFM1. The results show the ballistic stability of free T-AF28 and the complexes of T-AF28 / AFB1 and T-AF28 / AFM1. It can be seen that during the 50 ns equilibrium simulation, the free T-AF28 showed an upward trend between 0 and 10 ns, indicating that the conformation of free T-AF28 has undergone significant changes, such as the stem-loop of T-AF28 has undergone significant changes. Compared with free T-AF28, the RMSD curves of T-AF28 / AFB1 and T-AF28 / AFM1 complexes have a weaker upward trend within 10 ns than free T-AF28, indicating that the presence of AFB1 or AFM1 stabilizes the stem-loop conformation of T-AF28 to a certain extent. After 20 ns, the vibration of the three curves reached a stable state and the MD system reached equilibrium. In addition, compared with AF50, the RMSD curve of the optimized T-AF28 is relatively weak, indicating that T-AF28 can exist more stably in the solution.

[0036] like Figure 4 As shown in B, compared with T-AF28, the root mean square fluctuation (RMSF) curves of T-AF28-AFB1 and T-AF28-AFM1 complexes are lower than that of free T-AF28, indicating that the position fluctuation of T-AF28 bases in T-AF28-AFB1 complexes is smaller than that of T-AF28 itself. Therefore, we believe that the structure of T-AF28 itself is highly flexible and is not restricted by AFB1 or AFM1. It is worth mentioning that bases 5-24 corresponding to the T-AF28 sequence G5-C24 are located in the circular region of the secondary structure of T-AF28. The high flexibility of the circular region reveals the importance of the circular base in the binding process.

[0037] The radius of gyration is used to study the distribution characteristics of atoms in a system along a specific axis to evaluate the compactness or stability of the molecule. Figure 4 From the rotation radius curve of C, it can be seen that the amplitude of the rotation radius curve of the T-AF28 / AFB1 and T-AF28 / AFM1 complexes is weaker than that of free T-AF28, indicating that the structure of T-AF28 tends to stabilize the binding with AFB1 or AFM1. In addition, compared with AF50, the amplitude of the rotation radius curve of the optimized free T-AF28 is weaker, further indicating that T-AF28 can exist more stably in solution, which is consistent with the conclusion of the RMSD curve.

[0038] To further investigate the effect of non-covalent interactions between T-AF28 and AFB1 or AFM1, the molecular mechanics / Poisson-Boltzmann surface area (MM / PBSA) method was used to calculate the binding free energy. Figure 4 As shown in Figure F, the total interaction energy between T-AF28 and AFB1 is -37.44 kcal / mol, and the total interaction energy between T-AF28 and AFM1 is -34.96 kcal / mol, of which the van der Waals interaction energy (vdW) is -49.47 kcal / mol and -47.15 kcal / mol, respectively, which is much larger than the electrostatic interaction energy (-8.8 kcal / mol and -3.53 kcal / mol, respectively), indicating that vdW interaction is the main driving force for T-AF28 to recognize AFB1 and AFM1. In addition, the contribution of each base in the active center was also explored, and the results are shown in Figure 4D and 4E. It can be seen that the four cyclic bases (G5, T6, T23, and C24) of T-AF28 play an important role in recognizing AFB1, the vdW energy contribution of the active site of T-AF28 mainly comes from the bases G5, T23, and C24, and the electrostatic interaction energy contribution of T-AF28 mainly comes from the bases G5 and T6. The three cyclic bases (G5, T23, and C24) play an important role in recognizing AFM1, among which the vdW energy contribution of the active site of the aptamer mainly comes from the bases G5, T23, and C24, and the electrostatic interaction energy contribution sites of the aptamer mainly come from the bases T23 and C24.

[0039] like Figure 5 As shown in the figure, through molecular dynamics simulation, the three-dimensional structure of T-AF28 / aflatoxin analogs shows that T-AF28 can bind to aflatoxin analogs (AFB1, AFB2, AFM1 and AFM2), with a ladder-shaped binding domain and a consistent binding site, in which the aflatoxin analogs are located in the middle of the inverted ladder structure and captured by G5, T6 and T23, C24 bases. There are hydrogen bond interactions and π-π stacking between the G5 base and the target, hydrophobic interactions between the C24 base and the T6 base and the target, and strong hydrogen bond interactions between the T23 base and the target. The existence of these interactions leads to a strong affinity between T-AF28 and aflatoxin analogs. In addition, from the predicted secondary structure of T-AF28, it can be seen that the C4, G5 and T23 bases are all located near the stem loop, and the binding pocket of T-AF28 is located in the stem loop region, which plays an important role in the structural stability of the T-AF28 / aflatoxin analog complex.

[0040] 2. Construction of HCR / CuNPs-MoS 2 Detection of AFB1 and AFM1 by Ns aptamer sensing system A. Experimental Methods 1. Construction of HCR / CuNPs-MoS 2 Ns aptamer sensing system: T-AF28 and its complementary chain cDNA were dissolved in Tris-HCl buffer at pH 7.4, heated at 37°C for 60 min, and then cooled to room temperature. Subsequently, different concentrations of AFB1 or AFM1 were added, incubated at 37°C for 60 min, and then hairpin probes H1 and H2 were introduced. The nucleotide sequence of hairpin probe H1 is shown in the sequence list SEQ ID NO.5, and the nucleotide sequence of hairpin probe H2 is shown in the sequence list SEQ ID NO.6. The mixture was incubated at 37°C for 120 min, and copper ions and ascorbic acid were added, and incubated at room temperature for 10 min. Finally, the required amount of MoS 2 Ns was added to the sensing system, and the fluorescence spectrum was measured after standing for 5 min.

[0041] 2. Actual sample preparation: Crop samples, tea leaves, and edible oil were obtained from local supermarkets. Crop and tea samples were processed according to the previous method. 5 g corn or wheat samples were crushed and evenly mixed with a tissue grinder. Then, 20 mL of extraction solution (70% methanol / water solution) was added to the above powders. The mixture was vortexed for 20 min and centrifuged at 6000 rpm for 10 min. The supernatant was filtered with glass fiber filter paper to obtain a clear solution. Before extraction, different concentrations of AFB1 were added to the samples for recovery tests.

[0042] B. Experimental Results 1. Sensing strategy and feasibility discussion: The method described in the present invention shows the sensing strategy of AFB1, such as Figure 6 As shown in Figure 2, the strategy is based on the synergistic signal amplification of hybrid chain reaction (HCR) and 2D nanomaterials background reduction. Specifically, the sensing system is mainly composed of T-AF28 and its partially complementary oligonucleotide chain (cDNA), hairpin 1 (HP1), hairpin 2 (HP2) and MoS 2 Ns and in situ prepared CuNPs as fluorescent probes. T-AF28 can partially hybridize with cDNA through hydrogen bonds, while HP1 and HP2 are partially complementary to form a "hairpin" structure, which contains a stem of 25 base pairs, a loop of 5 nucleotides, and 6 unpaired bases as sticky ends. We noticed that the 3' end of HP1 can fully complement cDNA, and the 5' end of HP1 can fully complement the 3' end of HP2. After adding AFB1 or AFM1, the affinity of T-AF28 is higher than that of cDNA, and the released cDNA can act as an initiator to pair with the 3' end of HP1, triggering the melting of the "hairpin". The exposed chain in the hairpin structure HP1 is complementary to the 3' end chain in HP2, causing HP2 to melt. The 5' end chain of HP2 will hybridize with HP1 again after melting and repeated self-assembly to produce HCR. Thus, an ultra-long double-stranded DNA (ds-DNA) oligomer is formed, which can be used as an effective template for the in situ selective preparation of CuNPs. Based on MoS 2 The weak adsorption ability of Ns on ds-DNA can lead to the observation of strong and stable fluorescence emission of CuNPs.

[0043] In contrast, when AFB1 or AFM1 is absent, T-AF28 can capture cDNA to form sticky-tailed dsDNA, and no initiator can trigger the binding of HP1 and HP2 in the HCR system. HP1 and HP2 are adsorbed on MoS via sticky-end π-π stacking. 2 Ns surface. At the same time, the hairpin stem structure cannot be used as an effective template for the synthesis of CuNPs. CuNPs can only be synthesized in the T-AF28 / cDNA / DNA template and adsorbed on MoS 2Ns surface, through CuNPs and MoS 2 The fluorescence resonance energy transfer between MoS and Ns leads to the fluorescence quenching of CuNPs. 2 The introduction of Ns can adsorb the hairpin and short dsDNA of T-AF28 / cDNA, reduce the background interference signal, and improve the sensitivity and selectivity of the system to AFB1 and AFM. 2 The synergistic effect of Ns adsorption of single-stranded nucleotide chains and fluorescence quenching on CuNPs could obtain sensitive and selective detection of AFB1 and AFM1.

[0044] 2. Optimization of experimental conditions: In order to make the constructed aptamer sensing system have good detection performance, we optimized the MoS 2 Ns concentration, pH effect, Cu 2+ The experimental conditions such as ascorbic acid concentration, T-AF28 and cDNA dosage, H1 / H2 concentration, reaction time, etc. were optimized. The buffer medium has an important influence on the function of the aptamer. The effects of buffer type and system pH value on the detection of AFB1 and AFM1 were studied in detail. The results showed that the detection performance of the sensing system was best in MOPS buffer with a pH of 7.4 ( Figure 7 A. Figure 7 B).

[0045] Since MoS 2 Ns can adsorb oligonucleotides that do not participate in the HCR reaction in the sensing system and reduce background interference, so MoS 2 The amount of Ns is directly related to the detection of the target. Figure 8 As shown, with the MoS 2 As the concentration of Ns increases, the fluorescence quenching efficiency of the sensing system gradually increases. 2 When the Ns concentration was 50 μg / mL, the fluorescence quenching efficiency of the sensing system reached a plateau, indicating that in the absence of targets, the free oligonucleotides were almost completely quenched by MoS 2 Ns surface absorption. Therefore, further experiments were conducted using 50 μg / mL MoS 2 Ns.

[0046] When the captured oligonucleotides reach the target, the concentrations of T-AF28 and cDNA severely affect the detection of AFB1 and AFM1. Fig. 9As shown in A, with the increase in the concentration of T-AF28 and cDNA, the relative fluorescence change of the sensing system ((F-F0) / F0, where F and F0 are the fluorescence of the sensing system in the presence and absence of AFB1, respectively) gradually increased, reached a maximum value at a concentration of 0.5 μM, and then gradually decreased. Studies have shown that lower concentrations of T-AF28 / cDNA are not conducive to triggering HCR, while excessive T-AF28 / cDNA may cause significant background interference. The incubation time of the interaction between T-AF28 and AFB1 was also explored, and the results showed that their binding equilibrium can be reached at 60 min ( Fig.10 A). Therefore, 0.5 μM T-AF28 / cDNA and 60 min incubation time were set for further experiments.

[0047] As a signal reporter, CuNPs are composed of Cu in the dsDNA template generated by HCR. 2+ and ascorbic acid in situ synthesis, which plays an important role in the sensing system. Fig.11 As shown in A, in the range of 0.40~0.50mM, the relative change of the sensing system increases with the increase of Cu 2+ The concentration increases, and then increases with the increase of Cu 2+ The fluorescence of the sensor system decreases with further increase of concentration, indicating that low concentration of fluorescent probe cannot achieve high sensitivity for AFB1 detection, and too high concentration will cause concentration-effect fluorescence quenching. As a reducing agent, ascorbic acid can reduce copper ions to fluorescent copper ions. Subsequently, experiments on the dosage of ascorbic acid showed that within the range of 0.25-0.75 mM, the concentration of ascorbic acid had no significant effect on the fluorescence change of the sensor system ( Fig.11 B). Therefore, Cu 2+ The concentrations of HCl and ascorbic acid were fixed at 0.50 mM and 0.75 mM, respectively.

[0048] Next, the effect of hairpin H1 / H2 concentration on AFB1 detection was studied. The results showed that within the concentration range of 0.20-0.50 μM, the relative fluorescence change of the sensing system increased with the increase of H1 / H2 concentration, reached a peak at 0.50 μM, and then decreased ( Fig. 9 B). It can be seen that the medium dsDNA generated by HCR can provide a good template for the synthesis of CuNPs. Finally, the incubation time of the HCR process was discussed, such as Fig.10 As shown in B, the optimal incubation time for the hybridization chain reaction is 120 min, at which the performance of the sensing system is optimal. As mentioned above, the optimal experimental conditions for detecting AFB1 and AFM1 are: 50 μg / mL MoS 2 Ns, 0.5 μM T-AF28 / cDNA, 60 min incubation time, 0.50 mM Cu2+ and 0.75 mM ascorbic acid, pH 7.4 (MOPS), 120 min.

[0049] 3. Analysis and detection of AFB1 and AFM1: Under the optimal experimental conditions, HCR-MoS 2 Analytical performance of Ns / CuNPs aptasensor for detecting AFB1 and AFM1. Fig.12 As shown in the figure, with the increase of AFB1 concentration, the fluorescence intensity of the sensor system gradually increased. The fluorescence intensity of the sensor system showed a good linear relationship with the AFB1 concentration in the range of 0.031 ng / mL~46.84 ng / mL (y = 126.59x+362.73, R 2 = 0.992, and the detection limit (LOD) was calculated to be 0.0060 ng / mL. In addition, the sensor has the same detection performance for AFM1. The fluorescence of the sensing system also increases with the increase of AFM1 concentration, and has a good linear relationship with the AFM1 concentration in the range of 0.033 ng / mL to 32.83 ng / mL (y = 124.21x+361.15, R 2 =0.993), and the detection limit (LOD) was calculated to be 0.010 ng / mL. Compared with the reported detection results of AFB1 and AFM1 (Table 3), the constructed aptamer sensor has satisfactory detection performance, which may be due to the specific interaction between T-AF28 and AFB1 / AFM1 and the interaction between HCR and MoS 2 This is due to the synergistic effect of Ns signal amplification.

[0050] Table 3: Comparison of AFB1 and AFM1 test results reported previously 4. Selectivity and specificity: The constructed sensing system was used to measure the fluorescence response of potential coexisting analogs AFB1, AFM1, AFM2, as well as deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA) and other toxins to confirm the selectivity of the constructed sensing system. Fig.13As shown in A, the sensing system exhibited strong fluorescence responses to AFB1 and its analogs AFB2, AFM1, and AFM2, which was consistent with our molecular dynamics results. These experimental results clearly indicate that the T-AF28 we obtained may be a universal nucleic acid aptamer that can simultaneously recognize aflatoxin-like substances. Other toxins such as DON, ZEN, and OTA only caused a small fluorescence response in the sensing system. Further studies showed that the developed sensing system had good specificity for aflatoxin-like substances in the presence of other toxins. In addition, for actual samples, other possible coexisting amino acids and ions were explored, including Cys, Tyr, Gly, Trp, Ile, Glu, Ser, Phe, Val, Asp, Gln, Pro, Asn, His, Lys, Thr, Na + Mg 2+ , K + , Ca 2+ , Fe 2+ , Fe 3+ , Cu 2+ 、Zn 2+ , Mn 2+ 、Al 3+ Cr 3+ 、Cd 3+ , Hg 2+ and Ag + , to confirm the selectivity and specificity of the sensing system. Fig.13 B and Fig.13 As shown in C, the fluorescence response of the detection system to AFB1 and AFM1 was significantly higher than that of other potential co-existing interferences, indicating that the sensor system is specific to AFB1 and AFM1. In addition, the AFB1 and AFM1 mixture with co-interference of the control showed similar fluorescence response to AFB1 and AFM1, further indicating that the proposed method is specific and selective to AFB1 and AFM1.

[0051] 5. Method validation: To verify the feasibility of the proposed method in food samples, tea, edible oil, milk and other agricultural products were used as actual samples, and recovery experiments were carried out in the high, medium and low concentration linear ranges of AFB1 and AFM1. As shown in Tables 4 and 5, the sample recovery rates of AFB1 and AFM1 in actual samples were 85.2-117%, with relative standard deviations (RSD) less than 9.9%, and 85.5-117%, with RSD less than 9.9%, respectively. At the same time, compared with the detection results based on commercial ELISA kits, there was no significant difference in the detection performance of the two methods (the error was less than 10.0%), indicating that the developed HCR / CuNPs-MoS 2 The Ns sensing system can be used to detect AFB1 and AFM1 in actual samples.

[0052] Table 4: AFB 1 Recovery rate table in actual samples Table 5: AFM 1 Recovery rate table in actual samples Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0053] References: [1]Wang, Zhu, Chen, Zhu, Xiao, Yang, Chen, Rapid and visual detection of aflatoxin B1 in foodstuffs using aptamer / G-quadruplex DNAzyme probe with low background noise, Food Chemistry, 271(2019) 581-7. [2]Wang, Li, Zhao, A signal-on electrochemical aptasensor for rapid detection of aflatoxin B1 based on competition with complementary DNA, Biosensors and Bioelectronics, 144(2019) 111641. [3]Huang, Shu, Xiao, Pi, Li, Dai, Wang, Determination of aflatoxin B1in rice flour based on an enzyme-catalyzed Prussian blue probe, LWT, 162(2022) 113500. [4]Wu, Ali, Ouyang, Wang, Rong, Chen, Highly specific and sensitivedetection of aflatoxin B1 in food based on upconversion nanoparticles-blackphosphorus nanosheets aptasensor, Microchemical Journal, 171(2021) 106847. [5]Huertas-Pérez, Arroyo-Manzanares, Hitzler, Castro-Guerrero, Gámiz-Gracia, García-Campaña, Simple determination of aflatoxins in rice by ultra-high performance liquid chromatography coupled to chemical post-columnderivatization and fluorescence detection, Food Chemistry, 245(2018) 189-95. [6]Miró-Abella, Herrero, Canela, Arola, Borrull, Ras, Fontanals,Determination of mycotoxins in plant-based beverages using QuEChERS andliquid chromatography-tandem mass spectrometry, Food Chemistry, 229(2017)366-72. [7]Peltomaa, Abbas, Yli-Mattila, Lamminmäki, Single-stepnoncompetitive immunocomplex immunoassay for rapid aflatoxin detection, FoodChemistry, 392(2022) 133287。

Claims

1. A universal nucleic acid aptamer for broad-spectrum recognition of aflatoxins, characterized in that: The nucleic acid aptamer is a single-stranded DNA molecule sequence as shown in SEQ ID NO.3 in the nucleotide sequence table, and the specific sequence is GAACGTATTATCACTATGAGTCTCGTTC.

2. A method for screening and optimizing the universal nucleic acid aptamer for broad-spectrum recognition of aflatoxin according to claim 1 based on molecular dynamics, characterized in that: The secondary structure of the 50nt aflatoxin B1 aptamer AF50 was simulated using the online software Mfold web server. The structure consists of a 20-base ring structure, a 10-base neck structure, and 20 bases at both ends. Its nucleotide sequence is shown in SEQ ID NO.

1. The stem-loop structure is retained and the bases away from the two ends are deleted to obtain a truncated 28nt aptamer AF28. Its nucleotide sequence is shown in SEQ ID NO.

2. Then, RNA Composer software was used to obtain the tertiary structure of AF28. At the same time, combined with AutoDock 4.2.2 software, the binding pockets of AF28 with aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) were determined through molecular simulation docking program. By mutating some bases of AF28 in the stem and repeating the above molecular docking steps, the structurally stable optimized aptamer T-AF28 was obtained, and its nucleotide sequence is shown in SEQ ID NO.

3. Finally, LigPlot+ and PyMOL 2.3.3 software were used to visualize the molecular docking results and determine the core recognition regions of T-AF28 / AFB1 and AF28 / AFM1.

3. The method according to claim 2, characterized in that: The stem-loop structure was retained and the bases far from both ends were deleted to obtain the truncated 28nt aptamer AF28. The specific method was as follows: 5 bases at the 5′ end and 17 bases at the 3′ end of the stem-loop region far from AF50 were removed to obtain the aptamer AF28.

4. Use of the universal nucleic acid aptamer for broad-spectrum recognition of aflatoxins according to claim 1 in the preparation of products for detecting aflatoxins.

5. The use according to claim 4, characterized in that: The application of the universal nucleic acid aptamer for broad-spectrum recognition of aflatoxin in the preparation of reagents or drugs for detecting aflatoxin.

6. The use according to claim 5, characterized in that: The universal nucleic acid aptamer for broad-spectrum recognition of aflatoxins is used in the preparation of drugs or reagents for qualitative and / or quantitative detection of AFB1 and AFM1.

7. The use according to claim 5, characterized in that: The universal nucleic acid aptamer for broad-spectrum recognition of aflatoxins is used in the preparation of a kit for qualitative and / or quantitative detection of AFB1 and AFM1.

8. The use according to claim 5, characterized in that: The universal nucleic acid aptamer for broad-spectrum recognition of aflatoxins is used in the preparation of a biosensor for detecting AFB1 and AFM1.

9. The use according to claim 8, characterized in that: The biosensor is a biosensor for detecting AFB1 and AFM1 developed by a fluorescence method based on molybdenum disulfide nanosheet signal amplification technology; the biosensor is composed of T-AF28 and its partially complementary oligonucleotide chain cDNA, hairpin 1, i.e. HP1, hairpin 2, i.e. HP2 and MoS2Ns, and in-situ prepared CuNPs as fluorescent probes; the nucleotide sequence of the cDNA is shown in the sequence table SEQ ID NO.4; the nucleotide sequence of the hairpin probe H1 is shown in the sequence table SEQ ID NO.5, and the nucleotide sequence of the hairpin probe H2 is shown in the sequence table SEQ ID NO.

6.

10. The use according to claim 9, characterized in that: The biosensor is a HCR / CuNPs-MoS2Ns aptamer sensing system, specifically: 0.5 μM T-AF28 and its complementary chain cDNA are dissolved in MOPS buffer at pH 7.4, heated at 37°C for 60 minutes and then cooled to room temperature; different concentrations of AFB1 or AFM1 are added, incubated at 37°C for 60 minutes, and then 0.20-0.50 μM hairpin probes H1 and H2 are introduced, incubated at 37°C for 120 minutes, and then 0.50 mM copper ions and 0.75 mM ascorbic acid are added, and incubated at room temperature for 10 minutes; finally, 50 μg / mL MoS2Ns are added to the sensing system, and the fluorescence spectrum is measured after standing for 5 minutes.

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