Construction and application of histamine aptamer sensor based on sequence optimization

Abstract

The invention discloses an optimized aptamer for specifically recognizing histamine and application of the optimized aptamer, and belongs to the technical field of food safety analysis and detection. The optimized aptamer is characterized in that six dominant conformations of an H2 aptamer and histamine molecules are subjected to systematic docking analysis to obtain key binding characteristics of an aptamer-histamine compound, and a truncated aptamer H32 capable of specifically recognizing histamine is designed by deleting a non-functional region and retaining a core binding domain; compared with an original histamine H2 aptamer, the H32 aptamer has a Kd value of 2.5678 + / -1.1723 [mu] M, and the optimized aptamer has ideal affinity, good specificity and relatively low synthesis cost; the combination free energy of the H32 aptamer and histamine is remarkably reduced and is-32.1 kcal / mol, and the optimized aptamer has high structural stability and can effectively reduce the sensitivity of the aptamer to an application environment system.

Description

Technical Field

[0001] This invention relates to an optimized aptamer that specifically recognizes histamine and its application, belonging to the field of food safety analysis and detection technology. Background Technology

[0002] Histamine is one of the most toxic biogenic amines. When a person ingests products containing high concentrations of histamine, free histamine can rapidly enter the bloodstream through the gastrointestinal mucosa, bind to H1 receptors on vascular endothelial cells, increase capillary permeability, cause abnormal smooth muscle contraction, and thus trigger systemic pathological reactions. When histamine intake exceeds the individual's tolerance threshold, it can cause typical histamine poisoning symptoms, including facial flushing, headache, palpitations, and gastrointestinal discomfort. In terms of dosage, ingestion of more than 50 mg of histamine in healthy adults may induce mild poisoning, while more than 200 mg may cause severe poisoning. Histamine's thermal stability makes it difficult to destroy during conventional cooking; frying, baking, and other treatments can only eliminate 20% to 30% of the histamine content. There have been cases of histamine poisoning caused by consuming braised mackerel, with histamine levels reaching 464 mg / kg in retained food samples. Furthermore, histamine has a cumulative toxicity effect; long-term daily intake of 10-30 mg / kg of histamine can trigger chronic urticaria, migraines, and other abnormal conditions. WHO statistics show that histamine poisoning accounts for 28% of global seafood-related foodborne illnesses, with over 50,000 cases annually. Moreover, histamine contamination exhibits a "silent transmission" characteristic; contaminated raw fish can cross-contaminate the entire production line during processing. Experiments have confirmed that even before sensory indicators (such as odor and texture) show obvious signs of spoilage, histamine levels may already exceed safety limits by 3-5 times. Therefore, rapid and accurate detection of histamine levels in aquatic fish prone to histamine production is crucial for reducing food safety risks posed by histamine.

[0003] Currently, chromatography has become the preferred method for accurately determining histamine content in food due to its reliability. However, the inconvenience of the required analytical instruments, the difficulty in on-site monitoring, and the need for skilled technicians to perform pre-column derivatization limit its application in grassroots settings and areas requiring rapid detection. Electrophoresis, while highly sensitive, requires complex pretreatment steps and is subject to stringent environmental conditions. Electrochemical sensors offer advantages such as portability, high sensitivity, and low cost, making them suitable for rapid detection and real-time monitoring; however, their selectivity is susceptible to interference, their stability is insufficient, and environmental factors and complex sample matrices can significantly affect detection accuracy, limiting their widespread application in practical scenarios. In biometric detection technologies, enzyme-linked immunosorbent assay (ELISA) is highly sensitive but costly and complex; histamine dehydrogenase assays are highly specific but unstable; microbial sensing technology is suitable for batch screening but has a slow response; nucleic acid aptamers, as molecular recognition elements, can compensate for the shortcomings of the above methods due to their high specificity, high affinity, and ease of synthesis and modification. However, single-signal sensors are susceptible to interference from complex matrices, and redundant sequences in nucleic acid aptamer sequences increase synthesis costs and affect target binding efficiency.

[0004] Aptamers are single-stranded oligonucleotides that specifically bind to target substances, obtained by screening from artificially synthesized random nucleotide libraries using the exponential enrichment ligand evolution technique (SELEX). Aptamers can form specific three-dimensional conformations (hairpins, pseudoknots, convex rings, and G-tetramers, etc.) within a specific microenvironment, binding to the target substance with high affinity through intermolecular interactions such as spatial structure matching, hydrogen bonds, van der Waals forces, and electrostatic interactions. Compared with antibodies, aptamers have many advantages: (1) They are artificially synthesized, do not rely on animal immunization, and have small batch-to-batch variations; (2) They have good stability, can be stored for a long time, and are highly heat-resistant; (3) They have high affinity and high specificity, with aptamer Kd levels as low as nanomolar, effectively distinguishing structural analogs; (4) They are easy to modify, as labeling the ends of aptamers with chemical groups (such as FAM, ROX, FITC, and biotin) does not affect their affinity. Therefore, aptamers, as a novel type of recognition molecule, have unparalleled advantages in fields such as medical diagnosis, environmental monitoring, and food safety analysis, and have been integrated into various biosensing / analysis platforms.

[0005] Typically, aptamers screened directly using SELEX technology contain 70–130 nucleotides, including end primer regions and a middle random sequence region. However, not all nucleotides participate in the binding process between the aptamer and the target. Non-essential nucleotide sequence regions may form various secondary structures, thereby interfering with the binding of the aptamer and the target or reducing the conformational stability of the aptamer-target complex. Furthermore, longer sequences lead to lower yields and higher synthesis costs.

[0006] Therefore, this invention optimizes the aptamer sequence to address the problems of high synthesis cost and reduced target binding efficiency caused by redundant sequences in the nucleic acid aptamer sequence. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, this invention provides an optimized aptamer for specific histamine recognition and the construction and application of its sensor. The aim is to optimize the aptamer sequence by trimming and removing redundant structures while retaining balanced functional domains, thereby obtaining the shortest aptamer sequence and improving its histamine detection performance.

[0008] The first technical solution provided by the present invention is an optimized aptamer that specifically recognizes histamine, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] The optimized aptamer is H32, with the sequence 5'-CGTG TTGGT TGCGG TTCTT CCGAT CTGCTGTG-3'.

[0010] In some embodiments, the aptamer may be connected to a functional group or molecule.

[0011] Optionally, the functional group or molecule may be attached to the 5' end or 3' end of the aptamer.

[0012] In some embodiments, the functional group or molecule may be an isotope, a fluorescent marker, a chemiluminescent marker, a bioluminescent marker, an enzyme marker, a magnetic substance, biotin, an affinity ligand, and / or a thiol group.

[0013] The second technical solution provided by the present invention is a probe, wherein the probe is a substance obtained by labeling the optimized aptamer described in the first technical solution.

[0014] The third technical solution provided by the present invention is a cell containing the optimized aptamer described in the first technical solution or the probe described in the second technical solution.

[0015] In some embodiments, the cell may be an animal cell, a plant cell, or a microbial cell.

[0016] The fourth technical solution provided by the present invention is a biosensor containing the optimized aptamer described in the first technical solution or the probe described in the second technical solution.

[0017] The fifth technical solution provided by this invention is the application of the aptamer described in the first technical solution, the probe described in the second technical solution, the cell described in the third technical solution, or the biosensor described in the fourth technical solution in the detection of histamine.

[0018] The sixth technical solution provided by the present invention is a reagent or kit for detecting histamine, wherein the reagent or kit contains the aptamer described in the first technical solution or the probe described in the second technical solution.

[0019] The seventh technical solution provided by the present invention is a method for detecting histamine. The method includes labeling a reporter group on the aptamer described in the first technical solution, causing the aptamer labeled with the reporter group to interact with the sample to be tested, and detecting histamine by detecting the signal of the reporter group.

[0020] The eighth technical solution provided by this invention is the application of the aptamer described in the first technical solution, the probe described in the second technical solution, the cell described in the third technical solution, the biosensor described in the fourth technical solution, the reagent or kit described in the sixth technical solution, or the method described in the seventh technical solution in the food or biological field.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention uses aptamers to specifically recognize histamine. Compared with antibodies, it has the advantages of being able to be screened in vitro, having a short screening cycle, being easy to synthesize, being easy to label various functional groups and reporter molecules, and being stable and able to be stored and used for a long time. (2) The optimized aptamer of this invention is based on the systematic docking analysis of six advantageous conformations of the H2 aptamer with histamine molecules, which yielded the key binding characteristics of the aptamer-histamine complex (as shown in Table 1). Based on this, the H2 aptamer was systematically sequence optimized. By deleting non-functional regions (primer region, redundant stem-loop) and retaining the core binding domain, a truncated aptamer H32 that can specifically recognize histamine was designed. (3) Compared with the original histamine H2 aptamer obtained by screening, the Kd value of the H32 aptamer is 2.5678±1.1723uM. This optimized aptamer has ideal affinity, good specificity and low synthesis cost. (4) Compared with the original histamine H2 aptamer obtained by screening, the binding free energy of the H32 aptamer to histamine is significantly reduced to -32.1 kcal / mol. This optimized aptamer has strong structural stability and can effectively reduce the sensitivity of the aptamer to the application environment system. Attached Figure Description

[0022] Figure 1 The diagram shows the secondary and tertiary structures of the histamine aptamer; the AF diagram shows the secondary structure of the histamine aptamer, the af diagram shows the tertiary structure of the histamine aptamer, and ΔG below represents the Gibbs free energy of each structure.

[0023] Figure 2Analysis of the docking sites and interactions between the H2 aptamer and histamine; A and E are high-affinity conformations; D and F are low-affinity conformations; B and C are intermediate conformations among the six docking conformations, which are neither high-affinity nor low-affinity, and their binding properties are between high-affinity (A and E) and low-affinity (D and F).

[0024] Figure 3 Analysis of the docking sites and interactions between the H32 aptamer and histamine; A shows the secondary structure of the predicted optimized sequence, revealing that the Watson-Crick pairing pattern in the core stem-loop region is fully preserved, while the Loop 2 region is shortened, significantly improving structural rigidity; B reveals that the amino group (-NH2) of histamine forms hydrogen bonds with T18 (2.1 Å, 2.3 Å), and the imidazole ring forms hydrogen bonds with G13 and T15.

[0025] Figure 4 Analysis of the molecular dynamics optimized H2 and H32 aptamers and their interaction with histamine; A is the molecular dynamics optimized H2 aptamer, B is the molecular dynamics optimized H2 aptamer-histamine complex system showing three potential binding sites, C is the molecular dynamics optimized H32 aptamer, and D is the truncated H32 aptamer by removing redundant sequences.

[0026] Figure 5 A represents the binding energy between the aptamer and histamine and its hydrogen bonds; B represents the binding energy; C represents the hydrogen bonds that satisfy the bond length and bond angle; and D represents the hydrogen bonds that satisfy the bond angle.

[0027] Figure 6 The structural stability of the aptamer binding to histamine; A is the radius of gyration (Rg); B is RMSD; C is SASA.

[0028] Figure 7 A represents the affinity between the H2 aptamer and the H32 aptamer; B represents the binding performance of the H2 aptamer; and C represents the binding performance of the H32 aptamer.

[0029] Figure 8 A represents the specificity of H2 aptamers and H32 aptamers; B represents the specific recognition ability of H2 aptamers; C represents the specific recognition ability of H32 aptamers. Detailed Implementation

[0030] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0031] Test method: In the affinity assay, two aptamer solutions were added to two groups of histamine standard solutions of different concentrations, mixed, and incubated for 10 min. Then, 200 μL of AuNPs was added, and incubation continued for 50 min to promote the binding of the aptamer to histamine. Subsequently, 40 μL of 500 mM NaCl solution was added to adjust the ionic strength of the system. The UV-Vis absorption spectrum in the 200-800 nm range was measured, and the absorbance at 680 nm and 520 nm was recorded. The experiment was repeated three times, and the absorbance ratio at 680 nm to 520 nm (A680 / A520) was calculated to reflect the degree of binding between the aptamer and histamine.

[0032] For the specificity assay, several substances that may have similar structures or biological activities to histamine (tyramine, tryptamine, cadaverine, imidazole, spermidine, putrescine, and L-histidine) were selected as interfering agents to determine the specificity of H2 and H32 aptamers. After adding the aptamer solution to the interfering agent and incubating for 10 min, 200 μL of AuNPs was added, and incubation continued for 50 min. Subsequently, 40 μL of 500 mM NaCl solution was added, and the absorbance ratio (A680 / A520) at 680 nm and 520 nm was measured. The experiment was repeated three times, and all interfering agent solutions were prepared at a uniform 20 μM.

[0033] Example 1: Structural modeling of histamine H2 aptamer: Structural modeling of the histamine H2 aptamer was performed using the Mfold website. The sequence of the H2 aptamer (SEQ ID NO. 2: 5'-AGCTC CAGAA GATAA ATTAC AGGGA ACGTG TTGGT TGCGG TTCTT CCGAT CTGCTGTGTT CTCTA TCTGT GCCAT GCAAC TAGGA TACTA TGACC CCGG-3') was input, and the folding temperature was set to 37 °C. Na + With an ion concentration of 1.0 M and other default parameters, predict the secondary structure of the H2 aptamer. Export thermodynamic details, structural diagrams, and Vienna files. Analyze the secondary structure conformation using thermodynamic stability analysis. The formulas are as follows: ; in ΔG Represents the change in Gibbs free energy; ΔH Represents enthalpy change; ΔS represents entropy change; T represents temperature.

[0034] The sequence of the H2 aptamer and the secondary structure predicted in the Vienna file from the Mfold website were imported into the RNAComposer website to generate the initial tertiary structure. The generated tertiary structure was optimized using PyMOL software, correcting the phosphate backbone, adjusting the base stacking angle and geometry, and eliminating atomic conflicts through energy minimization. The histamine small molecule structure was downloaded from the PubChem database, and the initial conformation was optimized using Chem3D software, adjusting conformational rationality and performing energy minimization, before being saved as a mol2 structure. Hydrogenation was performed and Gasteiger charge was calculated using Auto Dock Tools software, and the structure was saved as a PDBQT file.

[0035] Secondary structure prediction based on the M fold website indicates that the H2 aptamer exists in six dominant conformations under physiological conditions. These six conformations share the common characteristic of containing 3-5 stem-loop structures. Figure 1 AF). According to conformational secondary structure analysis, all conformations contain a conserved stem-loop structure, formed by base pairing of C38, C39, G40 with C47, C48, G49, accompanied by two atypical wobbly pairs (AF). Figure 1 af). The base stacking angle of this region is 3.2 Å, and the helical twist angle is 34.5°, exhibiting typical B-type DNA conformation characteristics. A tertiary model shows that this stem-loop acts as the structural core, and the twist angle of its phosphate backbone forms a rigid-flexible alternating topological feature with the adjacent flexible region. Figure 1 (af). This structural feature not only enhances overall stability through base stacking but also provides a geometrically complementary recognition interface for target binding. The groove depth of this stem-loop is highly matched to the size of the histamine molecule, and may serve as the main binding site.

[0036] Example 2: Prediction of binding sites and molecular docking of histamine H2 aptamers: The aptamer surface mesh was generated based on the Lennard-Jones potential field, and potential binding sites of histamine on the aptamer surface were calculated. Polar hydrogen atoms were added to the H2 aptamer in Auto Dock Tools software to distribute the charge; the rigidity of the functional core region was preserved, while local flexibility was allowed in the remaining regions. The H2 aptamer was used as the acceptor, and the histamine molecule as the ligand. A docking box was set with the binding site coordinates predicted by RLDock software as the origin, and the mesh spacing was 0.375 Å. The Lamarckian genetic algorithm was used, with a maximum of 2.5 × 10⁻⁶ evaluations. 6 The Number of GA Runs was set to 50. The 10 conformational clusters with the lowest binding energies were extracted, and the average binding energy and the number of hydrogen bonds were calculated. The binding interface was visualized using PyMOL software, and key interactions were labeled.

[0037] Systematic docking analysis of six dominant conformations of the H2 aptamer with histamine molecules was performed using the Lamarckian genetic algorithm, revealing key binding characteristics of the aptamer-histamine complex (Table 1). The docking results showed that the binding free energy of all conformations was less than -4 kcal / mol (-4.035 to -4.623 kcal / mol), indicating a highly spontaneous thermodynamic binding process. Structures 1 and 5 exhibited the best binding performance, with their energy contribution primarily attributed to the synergistic effect of hydrogen bond energy and van der Waals interactions. Structures 1 and 5 showed optimal binding with histamine. Histamine molecules preferentially bound to the hydrophobic cavity below the stem-ring at the top of the H2 aptamer. Figure 2 ).

[0038] Table 1. Histamine aptamer molecular docking data

[0039] Example 3: Sequence optimization of histamine H2 aptamer: Based on the docking results of Example 2, the core binding domain and key bases were retained, and the H2 aptamer was optimized into the 32-base aptamer H32, whose nucleotide sequence is shown in SEQ ID NO.1 (5'-CGTGT TGGTT GCGGT TCTTC CGATCTGCTG TG -3').

[0040] Secondary structure prediction (Mfold) of the optimized sequence showed that the Watson-Crick pairing pattern in the core stem-loop region was fully preserved, while the Loop 2 region was shortened, significantly improving structural rigidity. Figure 3 A). Molecular docking of the H32 aptamer with histamine was performed to verify the aptamer's affinity and specificity. The results showed that its binding free energy with histamine was -4.16 kcal / mol, indicating that the H32 aptamer has good affinity for histamine. The structural integrity of the core stem-loop region is key to maintaining specific binding. The deletion of non-functional sequences did not disrupt the hydrogen bond network of the core binding domain, and the key interactions preserved the specific binding ability of histamine to the H32 aptamer. The amino group of histamine forms a hydrogen bond with T18, and the imidazole ring forms hydrogen bonds with G13 and T15. Figure 3(B) Removal of redundant stem-loops may eliminate potentially competitive binding sites, reduce the interference of conformational heterogeneity on target differentiation, and enhance the specific binding ability of the site to histamine. Reduced flexibility of Loop 2 decreases the conformational freedom of the unbound aptamer, leading to conformational entropy loss and increasing the thermodynamic driving force of the binding process. The rigid structure allows the binding domain to pre-approach the target binding conformation, lowering the binding energy barrier and potentially accelerating binding. The complete preservation of the core stem-loop region maintains 94% of the original binding energy and the hydrogen bond network, indicating that aptamer H32 can specifically bind to histamine.

[0041] Example 4: Modeling, molecular docking, and molecular dynamics simulation of the optimized histamine H32 aptamer with histamine. The optimized H32 aptamer sequence was modeled in the same manner as in Example 1, and docking was performed using the method described in Example 2. To further verify the accuracy of molecular docking and the dynamic stability of the binding of H2 and H32 aptamers to histamine, molecular dynamics simulations were used to systematically evaluate the affinity and binding specificity of the H2 and H32 aptamer-histamine complex systems. The initial aptamer structure was obtained through computational prediction and may contain some unreasonable conformations. The optimization process through molecular dynamics simulations can eliminate unreasonable conformations in the initial predicted structure, laying a good foundation for molecular dynamics simulations on longer time scales. After improving structural stability and thermodynamic rationality, it ensures that the aptamer structure changes in the actual buffer system are more closely approximated. Figure 4 A, C), thus better predicting its affinity and specificity for histamine binding. The molecularly dynamically optimized H2 aptamer-histamine complex system exhibits three potential binding sites (A, C). Figure 4 B). The truncated H32 aptamer eliminates two-thirds of the potential binding region at the site by removing redundant sequences, retaining only one core site (B). Figure 4 D). Molecular dynamics simulations show that the binding free energy of the H32 aptamer to histamine is significantly reduced to -32.1 kcal / mol, indicating that the optimized H32 aptamer has improved structural compactness and higher affinity.

[0042] To further investigate the interaction between the aptamer and histamine during motility and to assess the stability of the binding site to histamine, a 100 ns molecular dynamics simulation was performed on the complex. The dynamic stability of the specific binding of the aptamer to histamine was verified by dynamic binding energy, hydrogen bonds, radius of gyration, root mean square deviation, solvent, and surface area.

[0043] In the equilibrium phase of molecular dynamics simulations, the binding energy of the H2 complex system is around -7.5 kcal / mol, while the binding energy of the H32 complex system decreases to -10.5 kcal / mol. Figure 5The lower binding energy of the H32 complex system indicates that the H32 aptamer has a better affinity for histamine molecules.

[0044] The fluctuation in the number of hydrogen bonds in the H2 complex system indicates that there are more structural changes and relatively unstable hydrogen bonds in this system. In contrast, the stable hydrogen bonds in the H32 system reflect a stronger interaction between the aptamer and histamine. Figure 6 Furthermore, the H32 complex system has a greater number of hydrogen bonds and higher stability, which enhances the binding specificity of the aptamer to histamine.

[0045] The gyration radius Rg of the H2 aptamer-histamine complex system is stable between 2.2 and 2.4 nm, while that of the H32 aptamer-histamine complex system is stable between 1.3 and 1.5 nm. Figure 6 A). This indicates that the H32 aptamer has a more stable conformation and a more compact spatial structure when bound to histamine, which is superior to that of the H2 aptamer. The sequence optimization of the H32 aptamer effectively compresses the non-functional space and enhances the rigidity of the complex.

[0046] The RMSD value of the H2 aptamer-histamine complex system began to stabilize after about 20 ns, eventually settling between 1.2 and 1.5 nm. The H32 aptamer-histamine complex system exhibited less overall fluctuation, reaching equilibrium at approximately 10 ns, with an RMSD value between 0.5 and 0.7 nm. Figure 6 B). This indicates that the H32 aptamer-histamine complex exhibits faster equilibrium convergence and lower structural volatility during simulation, and its dynamic structural stability is significantly better than that of the H2 aptamer-histamine complex.

[0047] The SASA value of the H2 aptamer-histamine complex system was stable at 180 nm² / S² / N, while that of the H32 aptamer-histamine complex system was stable at 60 nm² / S² / N. Figure 6 C). This difference reflects that the H32 aptamer can reduce solvent exposure and enhance binding stability through the formation of a hydrophobic core. Furthermore, the hydrogen bond shielding effect also plays a similar role; the H32 aptamer forms stable hydrogen bonds with histamine, encapsulating the polar group within the complex and reducing competitive binding by water molecules. Figure 6 B). The lower SASA value indicates that the H32 aptamer binds tightly to histamine, has relatively weak hydrophobicity, has a smaller surface area exposed to the solvent, and the composite system is more stable.

[0048] Therefore, after sequence optimization, the H32 aptamer exhibits superior properties to the H2 aptamer in several key parameters. The H32 aptamer binds more tightly and stably to histamine, has a compact structure, stronger dynamic structural stability, lower hydrophobicity, less solvent exposure, and higher complex stability. It is reasonable to infer that the H32 aptamer has a good ability to recognize histamine.

[0049] Example 7: Affinity and specificity of histamine H2 and H32 aptamers: The optimized aptamer H32 and the original histamine aptamer H2 were subjected to affinity and specificity assays, as follows: 1. Determination of affinity: Two aptamer solutions were added to two groups of histamine standard solutions of different concentrations, respectively, and incubated for 10 min. 200 μL of AuNPs was added, and incubation continued for 50 min to promote aptamer-histamine binding. Subsequently, 40 μL of 500 mM NaCl solution was added to adjust the ionic strength of the system. The UV-Vis absorption spectra in the 200-800 nm range were measured, and the absorbance at 680 nm and 520 nm was recorded. The experiment was repeated three times, and the absorbance ratio at 680 nm to 520 nm (A680 / A520) was calculated to reflect the degree of aptamer-histamine binding. Nonlinear regression analysis was performed using formulas and Origin software to fit saturation curves, and the Kd value of the aptamer was determined to assess the aptamer affinity (e.g., ...). Figure 7 (As shown). The formula is as follows: (2); Where X represents the concentration of histamine; Y represents the absorbance ratio A680 / A520; Bmax represents the maximum absorbance ratio A680 / A520 when the aptamer is fully bound to the target; and Kd represents the dissociation constant.

[0050] In this embodiment, the affinity of the aptamer was determined using the optical method described above. The measured Kd value of the H2 aptamer was 47.1 μM. Figure 7 A), the Kd value of H32 is 2.6 μM ( Figure 7(B) The affinity of the H32 aptamer is 18-fold higher than that of the H2 aptamer. This clearly reflects the changing trend of the affinity between the H2 and H32 aptamers. Compared to H2, the binding between the H32 aptamer and histamine is more stable, the complex dissociates at a lower rate, and the binding is stronger. Furthermore, molecular dynamics simulations also show that the H32 aptamer has a superior binding energy and more stable hydrogen bonds than H2, indicating that the H32 aptamer has the ability to specifically recognize histamine. These results suggest that the predicted core binding region plays an important role in maintaining the stability of histamine binding, and the H32 aptamer obtained based on structural analysis and molecular docking optimization exhibits good affinity.

[0051] Table 2. Affinity data of the optimized aptamers

[0052] 2. Specificity determination: Several substances potentially structurally similar to histamine or possessing similar biological activities (tyramine, tryptophan, cadaverine, imidazole, spermidine, putrescine, L-histidine) were selected as interfering agents, and the specificity of H2 and H32 aptamers was determined. After adding the aptamer solution to the interfering agent and incubating for 10 min, 200 μL of AuNPs was added, and incubation continued for 50 min. Subsequently, 40 μL of 500 mM NaCl solution was added, and the absorbance ratio (A680 / A520) at 680 nm and 520 nm was measured. The experiment was repeated three times, and all interfering agent solutions were uniformly prepared to 20 μM (results are shown in the figure). Figure 8 (As shown).

[0053] like Figure 8 As shown, the H2 and H32 aptamers exhibit significantly higher specificity to histamine than other interfering molecules, indicating that the aptamers possess strong specificity for histamine. Furthermore, compared to the H2 aptamer, the H32 aptamer demonstrates higher signal-to-noise ratio and signal-to-background ratio, exhibiting superior specificity.

[0054] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An optimized aptamer that specifically recognizes histamine, characterized in that, The nucleotide sequence of the aptamer is shown in SEQ ID NO.

1.

2. The optimized aptamer according to claim 1, characterized in that, The aptamer may be connected to a functional group or molecule, which may be connected to the 5' end or the 3' end of the aptamer.

3. The optimized aptamer according to claim 2, characterized in that, The functional groups or molecules may be isotopes, fluorescent markers, chemiluminescent markers, bioluminescent markers, enzyme markers, magnetic substances, biotin, affinity ligands and / or thiol groups.

4. A probe, characterized in that, The probe is a substance obtained by labeling the optimized aptamer as described in any one of claims 1 to 3.

5. A cell containing the optimized aptamer of any one of claims 1 to 3 or the probe of claim 4.

6. A biosensor comprising the optimized aptamer of any one of claims 1 to 3 or the probe of claim 4.

7. The use of the aptamer according to any one of claims 1 to 3, the probe according to claim 4, the cell according to claim 5, or the biosensor according to claim 6 in the detection of histamine.

8. A reagent or kit for detecting histamine, characterized in that, The reagent or kit contains the aptamer as described in any one of claims 1 to 3 or the probe as described in claim 4.

9. A method for detecting histamine, characterized in that, The method includes labeling a reporter group onto an aptamer according to any one of claims 1 to 3, causing the aptamer labeled with the reporter group to interact with the sample to be tested, and detecting histamine by detecting the signal of the reporter group.

10. The use of the aptamer according to any one of claims 1 to 3, the probe according to claim 4, the cell according to claim 5, the biosensor according to claim 6, the reagent or kit according to claim 8, or the method according to claim 9 in the food or biological field.

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