Aptamer of peanut allergen arah 2, screening method and application

Abstract

This invention discloses aptamers, screening methods, and applications for the peanut allergen Ara h2, belonging to the field of molecular biology detection technology. This invention is the first to screen and obtain high-affinity and high-specificity DNA aptamers for Ara h2, filling a gap in the field of aptamer-specific recognition of this target. The obtained aptamers exhibit excellent affinity for Ara h2, and the Ara h2 biosensor designed using them is a dual-mode (colorimetric / fluorescent) aptamer sensor for Ara h2 constructed based on RCA-DNA enzyme cascade amplification technology. The LOD of the colorimetric method is 33 pg / mL, and the LOD of the fluorescence method is 8 pg / mL, demonstrating superior detection sensitivity compared to similar aptamer sensors. This invention, while possessing excellent specificity, provides a new platform for trace analysis of Ara h2 in food samples.

Description

Technical Field

[0001] This invention relates to aptamers, screening methods, and applications of peanut allergen Ara h 2, belonging to the field of molecular biological detection technology. Background Technology

[0002] Ara h 2, a peanut allergen, is the most potent allergen found in peanuts and belongs to the 2S albumin family. Its molecular structure contains four disulfide bonds, making it highly resistant to heat denaturation and pepsin digestion. It can pass intact through the gastrointestinal tract into the circulatory system, thus efficiently triggering an allergic reaction. Ara h 2 is recognized by serum IgE in over 90% of peanut allergy sufferers and is a key indicator of severe systemic anaphylactic reactions (including anaphylactic shock). Even extremely low intake can trigger life-threatening symptoms ranging from skin urticaria and respiratory laryngeal edema to sudden drops in blood pressure and loss of consciousness. Due to its extremely high allergenicity and stability, current processing methods are insufficient to completely eliminate its activity, making strict avoidance of peanut intake the only effective management strategy. Studies have shown that allergens not listed on ingredient labels have been detected in some products at concentrations as low as 1 mg per kilogram of food, due to unintentional cross-contamination during production, processing, or handling. This concentration is based on the detection limit of commercial testing kits, meaning lower concentrations are undetectable. However, even for the most sensitive individuals, extremely low doses (micrograms of protein) can trigger severe reactions. Currently, the main analytical techniques for detecting Ara h2 can be divided into protein-based methods and DNA-based methods. Protein methods rely on antigen-antibody binding, which, while specific, cannot bind to the amplification reaction, resulting in low sensitivity. Although DNA methods are highly sensitive, they are limited by expensive equipment and complex, time-consuming procedures. Therefore, convenient, highly sensitive, and specific methods for detecting and quantifying peanut allergens are crucial for ensuring food labeling compliance and strengthening consumer protection.

[0003] Aptamers are single-stranded DNA or RNA molecules. Due to their simple synthesis, high stability, good batch-to-batch consistency, ease of modification, and especially high specificity and sensitivity, they are considered ideal for allergen detection. More importantly, the introduction of aptamers helps in designing nucleic acid amplification protocols to improve sensitivity. In nucleic acid amplification methods, by designing and modifying aptamers, a single target signal can be combined with various amplification methods, thereby achieving signal conversion and amplification. Furthermore, nucleic acid aptamers that specifically recognize Ara h2 can eliminate interference from other proteins in the sample, enabling more accurate detection of Ara h2 levels and thus determining whether unlabeled peanut raw materials are present in the sample, helping peanut allergy sufferers avoid health risks. However, there are currently no publicly reported aptamers that specifically recognize Ara h2. Therefore, there is an urgent need to develop a high-affinity, high-specificity Ara h2 aptamer molecule, which would contribute to the construction of a sensitive biological detection platform and has clear scientific value and application prospects. Summary of the Invention

[0004] To address the shortcomings of the prior art, this invention aims to solve the problem of the lack of nucleic acid aptamers that can specifically recognize Ara h 2, and provides a high-affinity and specific Ara h 2 aptamer, its screening method, and a biosensor method for detecting Ara h 2 using the aptamer.

[0005] The first technical solution provided by the present invention is an aptamer that specifically binds to the peanut allergen Ara h 2, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0006] The second technical solution provided by this invention is a Capture-SELEX method for screening Ara h2-specific aptamers, specifically including the following steps: (1) ssDNA library fixation: Random ssDNA libraries with fixed regions are linked to streptavidin magnetic beads using biotin-labeled capture probes; (2) Target incubation and elution: The Ara h 2 protein solution was fully incubated with the immobilized ssDNA library to allow it to bind to the specific sequence; (3) PCR amplification and single-stranded DNA preparation: The enriched sequences that were eluted were amplified by PCR and the PCR products were purified. Single-stranded DNA was prepared by Lambda exonuclease and used as a library for the next round of screening. (4) High-throughput sequencing and candidate sequence screening: After the reverse screening in rounds 10 and 12, the enrichment rate was close to 51.7% in round 13, and the screening was terminated; the enriched library in round 13 was subjected to high-throughput sequencing, and candidate aptamers were determined based on the sequencing results for subsequent functional verification. (5) Affinity and specificity verification: The affinity and specificity of candidate aptamers were analyzed based on isothermal titration calorimetry (ITC) and fluorescence polarization method (FP). The sequence S2 with strong affinity and the best specificity was selected as the aptamer for the target. In some embodiments, in step (1), the nucleotide sequence of the ssDNA library is as follows: AATCAAACGCTAAGG-N10-GTGCACCCATTCTTG-N30-AAGCTTTGGTACCCGTATCGT.

[0007] In some embodiments, in step (1), the nucleotide sequence of the biotin-modified capture probe is as follows: 5′-Biotin-CAAGAATGGGTGCAC-3′.

[0008] In some embodiments, in step (3), the forward primer of the FAM label is 5′-FAM-AATCAAACGCTAAGG-3′, and the reverse primer is 5′-phosphate group-ACGATACGGGTACCAAGCTT-3′.

[0009] In some implementations, it also includes (6) binding mechanism study: using circular dichroism spectroscopy (CD) and molecular docking simulation, the conformational changes and binding regions of the aptamer binding with Ara h 2 are studied.

[0010] Using the above method, the present invention successfully screened Ara h 2 aptamers with high affinity and specificity, whose sequence is 5'-AATCAAACGCTAAGGTCAGCATCTTGTGCACCCATTCTTGGGTGTATTCTGTTTCTGTTTTGTATCTTTGAAGCTTGGTACCCGTATCGT-3'.

[0011] The third technical solution provided by the present invention is a colorimetric / fluorescence dual-mode aptamer sensor constructed using the aptamer described in the first technical solution.

[0012] In some embodiments, the colorimetric / fluorescence dual-mode aptamer sensor contains a complex formed by a DNase substrate chain and streptavidin magnetic beads, cDNA, and an RCA template, the nucleotide sequences of which are shown in SEQ and ID NO. 5-7.

[0013] The fourth technical solution provided by the present invention is a method for detecting peanut allergen Ara h2, wherein the method utilizes the colorimetric / fluorescence dual-mode aptamer sensor described in the third technical solution to detect the content of peanut allergen Ara h2.

[0014] In some embodiments, the method includes the following steps: (1) The aptamer S2 described in the first technical solution is incubated with cDNA to form an S2-cDNA complex; (2) After the S2-cDNA complex is mixed and incubated with the sample to be tested, it is added to the rolling circle amplification system to react and obtain the reactants. (3) Add the reactants to the complex formed by the DNase substrate chain and streptavidin magnetic beads and MgCl2, and incubate. After magnetic separation, obtain the supernatant containing fluorescent groups and magnetic beads connected to the substrate chain with exposed G-quadruplex regions. (4) The spectral signal values ​​of the supernatant and magnetic beads obtained in step (3) are detected by colorimetry and fluorescence method. The content of peanut allergen Ara h 2 in the sample is detected by the linear relationship between the signal value and the content of peanut allergen Ara h 2.

[0015] In some embodiments, the fluorescence method specifically involves measuring the fluorescence spectral signal value of the supernatant obtained in step (3) in the range of 500-650 nm at an excitation wavelength of 495 nm.

[0016] In some embodiments, the colorimetric method is specifically as follows: the magnetic beads from step (3) are dissolved in the reaction buffer, followed by the addition of heme, incubated at room temperature for 30 minutes, and then H2O2, ABTS and the reaction buffer are added. The mixture is placed in the dark and reacted at room temperature. After magnetic separation, the ultraviolet spectral signal value in the range of 400-500 nm is measured using a micro-volume cuvette.

[0017] In some embodiments, the method is as follows: aptamer S2 and cDNA are heated at 95°C for 5 minutes at a 1:1 molar ratio, and then slowly cooled to room temperature. The S2-cDNA complex is incubated with purified Ara h 2 in BB buffer at 37°C for 2 hours. Subsequently, 10 μL of the mixture is added to 50 μL of rolling circle amplification system (2.5 μL RCA template (10 μM), 2 μL T4 DNA ligase (5 U / μL) and 6 μL buffer, 4 μL phi29 DNA polymerase (10 U / μL) and 6 μL buffer, 2 μL dNTPs (25 mM) and 17.5 μL ultrapure water), and incubated at 37°C for 30 minutes. After the RCA reaction is complete, the temperature is adjusted to 65°C and held for 10 minutes to terminate the RCA reaction. Pretreated streptavidin magnetic bead-biotin substrate chain complex (MB-SS) and MgCl2 (50 mM) were then added, and the mixture was incubated at 37°C for 30 min. After magnetic separation, the fluorescence spectrum of the supernatant in the 500-650 nm range was measured at an excitation wavelength of 495 nm. The magnetic beads were dissolved in reaction buffer (10 mM Tris, 0.05% Triton X-100, 50 mM KCl, pH 7.4), heated at 95°C for 5 min, and then slowly cooled to room temperature. Heme (2 μM) was then added, and the mixture was incubated at room temperature for 30 min. After adding H2O2 (0.06 mM), ABTS (2 mM), and reaction buffer, the mixture was placed in the dark and reacted at room temperature for 10 min. After magnetic separation, the UV spectrum in the 400-500 nm range was measured using a microcuvette.

[0018] The fifth technical solution provided by the present invention is a reagent kit, which includes the aptamer described in the first technical solution, or the colorimetric / fluorescence dual-mode aptamer sensor described in the third technical solution.

[0019] The sixth technical solution provided by this invention is the application of the aptamer described in the first technical solution, or the colorimetric / fluorescence dual-mode aptamer sensor described in the third technical solution, or the reagent kit described in the fifth technical solution in the detection of peanut allergen Ara h 2.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides the first screening of high-affinity and high-specificity DNA aptamers targeting the peanut allergen (Ara h 2), filling a gap in aptamer recognition for this target. A reverse screening step was introduced during the screening process to effectively eliminate non-specific binding sequences to coexisting proteins, significantly improving aptamer selectivity. The binding ability and mechanism of the aptamers were systematically verified using multiple methods, including FP, ITC, CD, and molecular docking, ensuring the reliability and scientific validity of the screening results. The obtained aptamers exhibit excellent affinity for Ara h 2, with dissociation constants of 2.61 ± 1.27 μM and 18.91 ± 2.204 nM determined by ITC and FP methods, respectively, providing a molecular basis for the subsequent development of related recognition and detection technologies.

[0021] This invention utilizes the Ara h 2 biosensor designed with aptamer S2. It is a dual-mode (colorimetric / fluorescent) aptamer sensor for Ara h 2 constructed using RCA-DNase cascade amplification technology. The limit of detection (LOD) using the colorimetric method is 33 pg / mL, while the LOD using the fluorescence method is 8 pg / mL, which is superior to related peanut allergen biosensors. The results from the two modes corroborate each other, improving the accuracy of the method. This invention provides a new platform for trace analysis of Ara h 2 in food samples while exhibiting excellent sensitivity. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the screening principle of this method.

[0023] Figure 2 It is the secondary structure of the candidate aptamer.

[0024] Figure 3 This is an affinity analysis of ITC and FP for S1 and S2.

[0025] Figure 4 This is the specificity analysis of S1 and S2 using the FP method.

[0026] Figure 5 These are the CD spectra of S2 before and after binding to the target.

[0027] Figure 6 This is the result of S2 docking with the target HDOCK molecule.

[0028] Figure 7 This involves the preparation and characterization of MB-SS materials.

[0029] Figure 8 This is a schematic diagram of a colorimetric-fluorescence dual-mode aptamer sensor for Ara h 2.

[0030] Figure 9 These are the standard curves for colorimetric and fluorescence methods: A, UV-Vis absorption spectrum absorbance variation graph; B, linear relationship between the logarithm of Ara h2 concentration and absorbance; C, fluorescence intensity variation graph; D, linear relationship between the logarithm of Ara h2 concentration and absorbance.

[0031] Figure 10 This is a box plot showing the Ara h2 content in peanut milk determined by a dual-mode aptamer sensor, with concentrations of: A, 5 ng / mL; B, 10 ng / mL; C, 20 ng / mL. (t) 0.05,2 = 4.3027, no significant difference). Detailed Implementation

[0032] Reference Appendix Figures 1-10 The preferred embodiments of the present invention will be 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.

[0033] The reagents and raw materials used in this invention are as follows: Ara h 2 peanut allergen, purified in the laboratory; ready-to-use O′RangeRuler 10 bp DNA molecular weight standard kit; Dynabeads™ M-270 streptavidin magnetic beads purchased from Thermo Fisher Scientific; acrylamide / bisacrylamide (30% solution); N,N,N',N'-tetramethylethylenediamine (TEMED) purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; urea purchased from Maclean's Biochemical Technology Co., Lambda. 2,2'-Azobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydrogen peroxide (H2O2), sodium acetate (NaAc), acetic acid (HAc), tris(hydroxymethyl)aminomethane (Tris), bovine serum albumin (BSA), ovalbumin (OVA), ethylenediaminetetraacetic acid (EDTA), potassium dihydrogen phosphate (KH2PO4), potassium chloride (KCl), sodium chloride (NaCl), magnesium chloride (MgCl2), boric acid (H3BO3), ammonium persulfate, bromophenol blue, sucrose, and Tween X-100 were purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd. (Shanghai, China); exonuclease (5000 U / mL) and 10×Lambda exonuclease reaction buffer were purchased from New England BioLabs; Taq Plus DNA polymerase, dNTP mixture (25 mM solution), T4 DNA ligase (5 U / μL) and buffer, φ29 DNA polymerase (10 U / μL) and buffer, heme, and PCR purification kits were purchased from Sangon Biotech (Shanghai) Co., Ltd. All DNA strands were synthesized and purified (HPLC) by Sangon Biotech Co.

[0034] Example 1: Screening of Ara h 2 aptamers 1. ssDNA library fixation The randomized library (AATCAAACGCTAAGG-N10-GTGCACCCATTCTTG-N30-AAGCTTGGTACCCGTATCGT) and the biotin-modified capture probe (5′-biotin-CAAGAATGGGTGCAC-3′) were denatured in STE buffer at 95°C for 5 minutes and cooled to room temperature for 15 minutes at a final concentration of 1:1.5. The library was then incubated with pre-washed streptavidin-coated magnetic beads at 37°C and 200 rpm for 2 hours to immobilize the library on the magnetic bead surface.

[0035] 2. Target incubation and elution The magnetic beads immobilized with the ssDNA library were incubated with binding buffer containing 50 μg / mL Ara h 2 at 37°C and 200 r / min for 2 hours to allow the specific sequence to bind to the target. After magnetic separation, the supernatant was collected as a template for PCR amplification.

[0036] 3. PCR amplification and product purification The eluted enriched sequences were amplified by PCR in 50 μL reaction buffer containing 2 μL ssDNA template, 0.5 μL Taq DNA polymerase (5 U / μL), 5 μL 10× polymerase buffer, 0.5 μL of each primer (FAM-labeled forward primer (5′-FAM-AATCAAACGCTAAGG-3′), reverse primer (5′-phosphate-ACGATACGGGTACCAAGCTT-3′), 10 μM), 1 μL dNTP mixture (5 mM), and 40.5 μL sterile water. Amplification conditions were: 95 °C pre-denaturation for 5 min, 95 °C denaturation for 30 s, 58 °C annealing for 30 s, 72 °C extension for 2 min, and storage at 4 °C. The PCR products were purified to remove excess short-chain nucleic acids, dNTPs, ions, and salts from the amplification reaction system.

[0037] 4. Single-chain preparation Lambda exonuclease and its buffer were added to the purified PCR product and mixed thoroughly. The mixture was incubated at 37°C until the exonuclease completely cleaved the phosphate-modified reversed nucleic acid strand. After complete digestion, the reaction was terminated at 75°C for 10 min. The digested product was identified using an 8% denaturing polyacrylamide gel electrophoresis. Finally, the ssDNA was purified by ethanol precipitation and collected as the next library.

[0038] 5. Enrichment rate monitoring and high-throughput sequencing In each round of screening, enrichment efficiency was monitored using fluorescent labeling. In rounds 10 and 12, a reverse screening was performed using an equimolar mixture of Ara h 6 and Ara h 1 to eliminate candidate sequences capable of cross-binding with non-target allergens, thereby improving the specific recognition ability of the obtained aptamers for Ara h 2. Ara h 6 and Ara h 2 belong to the 2S albumin family and are highly homologous in amino acid sequence and spatial conformation. Ara h 6 is one of the allergens in peanuts with the strongest cross-reactivity with Ara h 2. Meanwhile, Ara h 1 is one of the most abundant major allergens in peanuts, belonging to the 7S globulin family, although its structure does not show significant similarity to Ara h 2. However, in complex food matrices, Ara h 1 often coexists with Ara h 2, and non-specific aptamers may bind to it through charge or hydrophobic interactions; therefore, both must be excluded. Using equimolar mixing ensures that all non-target proteins are at equal competitive concentrations in the reverse screening system, avoiding screening bias due to concentration differences and thus comprehensively removing non-specific aptamers targeting Ara h1 and Ara h6. This reverse screening strategy effectively reduces the false positive rate and ensures the accuracy of Ara h2 detection in complex food matrices. Therefore, using an equimolar mixture of Ara h6 and Ara h1 for reverse screening can effectively remove interfering background in food matrices and improve detection specificity.

[0039] The enrichment rate approached 51.7% in the 13th round, terminating the screening process. The enriched library from the 13th round was subjected to high-throughput sequencing. Four candidate aptamers, S1, S2, S3, and S4 (nucleotide sequences shown as SEQ ID NO. 1–4), were selected based on secondary structure, free energy (ΔG), and sequence frequency for affinity and specificity experiments (Table 1). Figure 2 ).

[0040] Table 1. Basic information on candidate aptamer sequences

[0041] Example 2: Affinity and Specificity Analysis of Candidate Aptamers 1. Assess aptamer binding capacity by isothermal titration calorimetry (ITC). Ara h 2 and candidate aptamers (S1–S4) were dissolved separately in 10 mM Tris-HCl buffer. 70 μL of a 100 μM candidate aptamer solution was loaded into a syringe, and 300 μL of a 6 μM Ara h 2 solution was added to the sample cell. The first injection was 0.4 μL, followed by 2 μL injections, for a total of 18 injections at 150 s intervals, with stirring at 750 rpm to ensure complete mixing after each injection. The corrected isothermal binding curves were fitted using the unit point binding model in the MicroCal PEAQ-ITC analysis software to obtain binding affinity and thermodynamic parameters.

[0042] 2. Verify affinity and specificity using fluorescence polarization (FP) method.

[0043] Ara h 2 solutions (10–200 nM) were incubated with 5'-FAM-labeled candidate aptamers (5 nM) in BB buffer at 37 °C for 2 hours in the dark. After incubation, fluorescence polarization was measured using a multi-functional plate reader. A blank control was performed using BB buffer instead of Ara h 2 under the same conditions. The change in fluorescence polarization (ΔFP) was calculated as ΔFP = FP1 – FP0, where FP0 is the polarization of the control and FP1 is the polarization of the test group. The dissociation constant (Kd) was determined by nonlinear fitting using GraphPad Prism 8.0 software. To assess specificity, Ara h 2 was replaced with structural analogs or potentially coexisting proteins, including Ara h 6, Ara h 1, β-conglycinin, glycinin, casein, bovine serum albumin, and chicken ovalbumin, under the same conditions.

[0044] 3. Select candidate sequences with high affinity and good specificity as the best aptamers for this target. The affinity results determined by the ITC and FP methods are consistent. (See Table 2 and...) Figure 3 As shown, S2 has a lower dissociation constant and exhibits higher affinity for the target compared to other candidate aptamers. Specificity analysis results for S1 and S2 are as follows... Figure 4 As shown, both aptamers clearly distinguished between Ara h 2 and non-target proteins, confirming their specificity. The relative binding rates of S2 to Ara h 6, Arah 1, β-conglycinin, glycinin, casein, bovine serum albumin, and chicken ovalbumin were all less than 20%. This indicates that aptamer S2 has low cross-reactivity with coexisting or structurally similar molecules. Therefore, S2, which specifically binds to Ara h 2 and has high affinity, was selected for further investigation.

[0045] Table 2 Results of affinity determination for candidate aptamers

[0046] Example 3: Analysis of the optimal aptamer-target binding mechanism 1. Circular dichroism (CD) spectroscopy analysis of conformational changes after aptamer binding. A mixture of 4 μM Ara h 2 and 5 μM S2 aptamers was prepared in BB buffer and incubated at 37 °C and 200 rpm for 2 h. Baseline signals in BB buffer and 4 μM Ara h 2 in BB buffer were recorded and subtracted from the final spectrum. CD spectra were recorded in the 220–320 nm range at room temperature. As shown in Figure 5, the CD signal at 276 nm decreased after S2 bound to Ara h 2. This may be because the original secondary structure of the aptamer needs to unwind or unfold during interaction with the target, thus reducing base stacking. Conversely, the circular dichroism signal at 245 nm increased, possibly because the more typical helical twist angle enhanced the chiral environment.

[0047] 2. Analysis of aptamer-target binding sites based on molecular docking The protein structure of Ara h 2 was obtained from the RCSB PDB database (https: / / www.rcsb.org / , PDB ID: 8DB4) at a resolution of 2.30 Å. Water molecules were removed prior to docking. To construct the 3D structure of the aptamer, its secondary structure and Vienna format were first predicted using the Mfold online web server (http: / / unafold.rna.albany.edu / ?q=mfold), and then the 3D structure with the optimal predicted energy was generated using the RNAcomposer online tool. The final 3D conformation was obtained by mutating U bases to T bases. Molecular docking experiments were performed using HDOCK Server, and the optimal docking model was selected by docking score selection. The docking results are shown in Figure 6. There are 15 binding sites on S2 (C-49, T-50, G-51, T-52, T-53, T-54, C-55, T-56, A-64, C-66, T-67, T-69, G-70, A-72, G-73), which bind to Ara h 2 mainly through non-covalent interactions such as hydrogen bonds, salt bridges, and hydrophobic interactions.

[0048] Example 4 Construction of a colorimetric / fluorescence dual-mode aptamer sensor 1. Preparation and characterization of MB-SS The design method for the DNase substrate chain (SS) is described in Table 3. Lyophilized SS was dissolved in DEPC water, denatured at 95°C for 5 minutes, and then slowly cooled to room temperature. Streptavidin beads (MB, 200 μL, 10 mg / mL) were washed sequentially with PBS and STE buffer via magnetic separation to obtain pretreated MB. The dissolved SS was diluted to 1 μM with STE buffer, and 200 μL of this solution was incubated with the pretreated MB at room temperature for 1 hour with shaking at 450 rpm to form the MB-SS complex. This complex was stored at 4°C until use.

[0049] The coupling reaction of MB-SS was verified by zeta potential. After coupling, the zeta potential changed from -12.30 mV to -41.35 mV. Figure 7 This significant decrease is attributed to the negatively charged phosphate backbone of the nucleic acid.

[0050] 2. Construction of a colorimetric-fluorescence dual-mode aptamer sensor Figure 8 The mechanism of action of the Ara h 2 colorimetric / fluorescence dual-mode optical sensor is illustrated. All sequences used in the optical sensor are listed in Table 3 (nucleotide sequences are shown in SEQ ID NOs 5-7, respectively). This invention designs a cDNA trigger sequence complementary to the S2 region of the aptamer to promote linear RCA template circularization and initiate the RCA reaction. In the absence of Ara h 2, the aptamer hybridizes with the cDNA, thereby preventing its release. In the presence of Ara h 2, the S2 region preferentially binds to the target, thereby releasing the cDNA and triggering the RCA reaction. Using the pre-designed RCA template, in Mg... 2+ In the presence of [a specific substance], the RCA product acquires DNase cleavage activity, releasing the 3' end of the FAM-tagged substrate strand immobilized on the magnetic beads. This exposes the G-quadruplex region, which is located at [a specific location]. + In its presence, it binds to heme and exhibits peroxidase-like activity, catalyzing the oxidation of ABTS, thereby producing UV absorption at 417 nm. This dual-mode platform enables sensitive and specific detection of Arah 2.

[0051] The specific procedures are as follows: First, the aptamer S2 and cDNA were heated at 95°C for 5 minutes at a 1:1 molar ratio, and then slowly cooled to room temperature. The S2-cDNA complex was incubated with purified Ara h 2 in BB buffer at 37°C for 2 hours. Subsequently, 10 μL of the mixture was added to 50 μL of rolling circle amplification system (2.5 μL RCA template (10 μM), 2 μL T4 DNA ligase (5 U / μL) and 6 μL buffer, 4 μL phi29 DNA polymerase (10 U / μL) and 6 μL buffer, 2 μL dNTPs (25 mM) and 17.5 μL ultrapure water), and incubated at 37°C for 30 minutes. After the RCA reaction was complete, the temperature was adjusted to 65°C and held for 10 minutes to terminate the RCA reaction. Pretreated streptavidin magnetic bead-biotin substrate chain complex (MB-SS) and MgCl2 (50 mM) were then added, and the mixture was incubated at 37°C for 30 min. After magnetic separation, the fluorescence spectrum of the supernatant in the 500–650 nm range was measured at an excitation wavelength of 495 nm. The magnetic beads were dissolved in reaction buffer (10 mM Tris, 0.05% Triton X-100, 50 mM KCl, pH 7.4), heated at 95°C for 5 min, and then slowly cooled to room temperature. Heme (2 μM) was then added, and the mixture was incubated at room temperature for 30 min. After adding H2O2 (0.06 mM), ABTS (2 mM), and reaction buffer, the mixture was placed in the dark and reacted at room temperature for 10 min. After magnetic separation, the UV spectrum in the 400–500 nm range was measured using a microcuvette. All measurements were repeated three times.

[0052] 3. Establishment of the standard curve Under optimal conditions, the analytical performance of the dual-mode aptamer sensor used to detect Ara h2 was evaluated. Figure 9 As shown in Figure C, the fluorescence intensity of the system gradually increased with increasing Ara h2 concentration. A strong linear relationship was established between the fluorescence intensity difference ΔF at 520 nm and the logarithm of the Ara h2 concentration within the range of 0.2 ng / mL to 80 ng / mL. The corresponding calibration curve (…) Figure 9 D) The equation ΔF = 275.012 + 314.558 log C (ng / mL) was obtained, with a correlation coefficient R. 2 The value was 0.991. Simultaneously, with increasing Ara h2 concentration, the absorbance measured at 417 nm also increased. Within the concentration range of 2 ng / mL to 320 ng / mL, a linear relationship was found between the absorbance difference ΔA at 417 nm and the logarithm of the analyte concentration. Figure 9(A, 8B), the equation ΔA = -0.036 + 0.310 log C (ng / mL) describes the result (R² = 0.993). Using the 3σ / k standard, the limit of detection (LOD) was 8 pg / mL in fluorescence mode and 33 pg / mL in colorimetric mode. Compared with previously reported methods, the developed aptamer sensor exhibits superior sensitivity and overall analytical performance.

[0053] Table 3 Sequence information used in the dual-mode aptamer sensor

[0054] Example 5: Actual Sample Testing Multiple food samples purchased from local supermarkets—peanut milk, chocolate cake, and plant-based soy meat—were selected as actual samples for spiked recovery experiments. The spiked recovery detection of the pretreated actual samples was performed using the aforementioned colorimetric / fluorescence dual-mode aptamer sensing method and the Ara h 2 enzyme-linked immunosorbent assay kit to verify the practicality of the method of the present invention.

[0055] The pretreatment steps for the real samples were as follows: After homogenizing the sample in a grinder, 1 gram of sample was dissolved in 10 mL of Tris-HCl buffer (50 mM, pH 7.2) and homogenized again. The sample was then centrifuged at 9,000 rpm / min for 10 minutes at 4°C. Before analysis, the supernatant was diluted tenfold with Tris-HCl buffer. For the chocolate sample, it was frozen at -20°C before homogenization, and 1 gram of skim milk powder was added before extraction. Skim milk powder contains a large amount of casein, which can bind to the high levels of phenolic compounds (such as tannins) in the matrix. The Ara h2 concentration was determined using this developed method and ELISA kit, and the corresponding recovery rate was calculated. Background values ​​were determined using the ELISA kit.

[0056] As shown in Table 4 and Figure 10 As shown, there was no significant difference between the results obtained by colorimetric and fluorescence modes, and there was also no significant difference from the results obtained by the ELISA kit. The results indicate that the colorimetric / fluorescence dual-mode aptamer sensing method constructed in this invention can be successfully applied to the analysis of Ara h2 in food samples.

[0057] Table 4. Detection results and recovery rates of Ara h 2 in plant-based soy meat, chocolate cake, and peanut milk (n=3)

[0058] 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 aptamer that specifically binds to the peanut allergen Ara h2, characterized in that, The nucleotide sequence of the aptamer is shown in SEQ ID NO.

2.

2. A Capture-SELEX method for screening Ara h2-specific aptamers, characterized in that, Specifically, the following steps are included: (1) ssDNA library fixation: Random ssDNA libraries with fixed regions are linked to streptavidin magnetic beads using biotin-labeled capture probes; (2) Target incubation and elution: The Ara h 2 protein solution was fully incubated with the immobilized ssDNA library to allow it to bind to the specific sequence; (3) PCR amplification and single-stranded DNA preparation: The enriched sequences that were eluted were amplified by PCR and the PCR products were purified. Single-stranded DNA was prepared by Lambda exonuclease and used as a library for the next round of screening. (4) High-throughput sequencing and candidate sequence screening: After the reverse screening in rounds 10 and 12, the enrichment rate was close to 51.7% in round 13, and the screening was terminated; the enriched library in round 13 was subjected to high-throughput sequencing, and candidate aptamers were determined based on the sequencing results for subsequent functional verification. (5) Affinity and specificity verification: The affinity and specificity of candidate aptamers were analyzed based on isothermal titration calorimetry and fluorescence polarization method, and the sequence with strong affinity and the best specificity was selected as the aptamer of the target.

3. The method according to claim 2, characterized in that, In step (1), the nucleotide sequence of the ssDNA library is as follows: AATCAAACGCTAAGG-N10-GTGCACCCATTCTTG-N30-AAGCTTGGTACCCGTATCGT; The nucleotide sequence of the biotin-modified capture probe is shown below: 5′-Biotin-CAAGAATGGGTGCAC-3′; In step (3), the forward primer for FAM labeling is 5′-FAM-AATCAAACGCTAAGG-3′, and the reverse primer is 5′-phosphate group-ACGATACGGGTACCAAGCTT-3′.

4. A colorimetric / fluorescence dual-mode aptamer sensor constructed using the aptamer of claim 1.

5. The colorimetric / fluorescence dual-mode aptamer sensor according to claim 4, characterized in that, The colorimetric / fluorescence dual-mode aptamer sensor contains a complex formed by a DNase substrate chain and streptavidin magnetic beads, cDNA, and an RCA template, the nucleotide sequences of which are shown in SEQ and ID NO. 5-7.

6. A method for detecting peanut allergen Ara h2, characterized in that, The method involves using the colorimetric / fluorescence dual-mode aptamer sensor described in any one of claims 4 to 5 to detect the content of peanut allergen Ara h 2.

7. The method according to claim 6, characterized in that, The method includes the following steps: (1) The aptamer of claim 1 is incubated with cDNA to form an S2-cDNA complex; (2) After the S2-cDNA complex is mixed and incubated with the sample to be tested, it is added to the rolling circle amplification system to react and obtain the reactants. (3) Add the reactants to the complex formed by the DNase substrate chain and streptavidin magnetic beads and MgCl2, and incubate. After magnetic separation, obtain the supernatant containing fluorescent groups and magnetic beads connected to the substrate chain with exposed G-quadruplex regions. (4) The spectra of the supernatant and magnetic beads obtained in step (3) are detected by colorimetric and fluorescence methods. The content of peanut allergen Ara h 2 in the sample is detected by the linear relationship between the signal value and the content of peanut allergen Ara h 2.

8. The method according to claim 7, characterized in that, The fluorescence method specifically involves measuring the fluorescence spectral signal value of the supernatant obtained in step (3) in the range of 500-650 nm at an excitation wavelength of 495 nm; the colorimetric method specifically involves dissolving the magnetic beads from step (3) in a reaction buffer, then adding heme, incubating at room temperature for 30 minutes, adding H2O2, ABTS and the reaction buffer, placing the mixture in the dark for room temperature reaction, separating it by magnetic force, and then measuring the ultraviolet spectral signal value in the range of 400-500 nm using a micro-volume cuvette.

9. A reagent kit, characterized in that, The kit includes the aptamer as described in claim 1, or the colorimetric / fluorescence dual-mode aptamer sensor as described in any one of claims 4 to 5.

10. The use of the aptamer of claim 1, or the colorimetric / fluorescence dual-mode aptamer sensor of any one of claims 4-5, or the kit of claim 9 in the detection of peanut allergen Ara h 2.

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