A method for constructing a dual-identification electrochemical biosensor and application thereof in detecting 3-amino phenetol in animal food

Abstract

The application discloses a construction method of a double-identification electrochemical biosensor and application of the double-identification electrochemical biosensor in detection of 3-aminoanisole in animal food, and relates to the technical field of food safety detection. Firstly, 3-amino-5-ethoxybenzoic acid is used as a hapten, a complete antigen is prepared by applying a carbodiimide method, and an animal is immunized to obtain a specific monoclonal antibody; meanwhile, a 3-aminoanisole nucleic acid aptamer is obtained through SELEX screening; an antibody-aptamer conjugate is prepared by using a biotin-streptavidin method, and is fixed on the surface of a gold electrode modified by a nano-sensitizing material to construct a double-identification electrochemical sensing interface, and an electrochemical sensing detection method is established. The electrochemical sensor developed in the application has the advantages of low detection limit, wide linear range, high precision and accuracy, strong specificity, good anti-interference ability, fast response speed, simple and rapid operation and the like, and can be used for trace detection and risk analysis of 3-aminoanisole in animal food.

Description

Technical Field

[0001] This invention relates to the field of food safety testing, and in particular to a method for constructing a dual-recognition electrochemical biosensor and its application in detecting 3-aminophenethyl ether in animal-derived foods. Background Technology

[0002] Ethoxybenzone, a highly effective anticoccidial drug, is widely used in poultry farming. However, it is slowly metabolized in food animals, leaving residues in tissues, particularly accumulating in internal organs such as the liver and kidneys, posing a clear food safety risk. Both my country's national food safety standard, "Maximum Residue Limits for Veterinary Drugs in Food" (GB 31650-2019), and the US Food and Drug Administration (21 CFR 556.260) clearly stipulate that the residue marker for ethoxybenzone is 3-aminophenethyl ether, also known as m-aminophenethyl ether. The maximum residue limit for 3-aminophenethyl ether in chicken is 500 μg / kg, and for chicken liver and kidney it is 1500 μg / kg.

[0003] Establishing efficient and accurate detection methods is essential for ensuring the safety of animal-derived food and consumer health. Currently, detection methods mainly include high-performance liquid chromatography (HPLC), chromatography-mass spectrometry (GC-MS), and immunoassay. While HPLC and GC-MS offer advantages such as high sensitivity and accuracy, they also have inherent limitations, including long detection cycles, expensive equipment, specialized operation, and complex sample preparation, making them unsuitable for rapid, large-scale on-site screening. Immunoassays, such as enzyme-linked immunosorbent assay (ELISA), offer advantages such as speed, simplicity, and high throughput, making them suitable for initial screening of large batches of samples. However, this method relies on highly specific antibodies. 3-Aminophene ether, as a small molecule hapten, is not immunogenic in itself and must be conjugated with a carrier protein to prepare an artificial complete antigen before immunizing animals to obtain specific antibodies. This process is technically complex and time-consuming, and the resulting traditional antibodies still have limitations in terms of stability, batch-to-batch consistency, and affinity.

[0004] Electrochemical biosensors have shown great potential in the field of rapid detection due to their advantages of simple equipment, low cost, fast response, and ease of miniaturization and automation. However, traditional electrochemical biosensors based on biological antibodies still have significant shortcomings in practical applications: the sensor interface is easily contaminated by complex sample matrices, and non-specific adsorption is serious, resulting in poor signal stability and low reproducibility, making it difficult to meet the requirements for long-term stable detection of complex samples. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing an electrochemical antibody-aptamer dual recognition sensor based on nanomaterial signal amplification, so as to solve the problems of complex operation, poor stability and low sensitivity of existing detection methods.

[0006] Another object of the present invention is to provide an electrochemical antibody-aptamer dual recognition sensor prepared by the above-described construction method.

[0007] Another object of the present invention is to provide an application of the above-mentioned sensor for detecting 3-aminophenethyl ether in animal-derived foods.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for constructing a dual-recognition electrochemical biosensor, comprising the following steps: Step 1: Using 3-amino-5-ethoxybenzoic acid as a hapten, the 3-amino-5-ethoxybenzoic acid hapten was obtained after ester hydrolysis and purification. Step 2: The hapten obtained in Step 1 is sequentially protected with amino groups, added to a spacer arm, coupled with a carrier protein, deprotected, and purified to obtain the complete antigen. Step 3: Using the complete antigen obtained in Step 2 as an immunogen, immunize the animals to prepare specific antibodies; the antibodies are monoclonal antibodies or polyclonal antibodies. Step 4: Using 3-aminophenethyl ether as a target, nucleic acid aptamers that specifically recognize 3-aminophenethyl ether are screened using SELEX technology. The screening includes forward screening and reverse screening steps. Step 5: The antibody obtained in step 3 is conjugated with the aptamer obtained in step 4 using the biotin-streptavidin method to prepare antibody-aptamer conjugates. Step 6: Modify the surface of the gold electrode with nano-sensitizing material to form a signal amplification substrate; add bovine serum albumin solution for the first blocking; dilute the antibody-aptamer conjugate obtained in step 5 with phosphate buffer and add it to the blocked substrate surface for incubation; add bovine serum albumin solution again for the second blocking; after interface activation, the dual-recognition electrochemical sensing interface is constructed.

[0009] As a preferred embodiment, in step 3, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:9, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:3, SEQ ID NO:7, or SEQ ID NO:11; the nucleotide coding sequences of the heavy chain variable region are shown as SEQ ID NO:2, SEQ ID NO:6, or SEQ ID NO:10, and the nucleotide coding sequences of the light chain variable region are shown as SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:12, respectively; the PCR amplification primers for the monoclonal antibody variable region gene are selected from any one or more pairs of the following primers: the nucleotide sequences of the upstream primers VH-F1 to VH-F10 of the heavy chain variable region are shown as SEQ ID NO:23 to SEQ ID NO:32, respectively; the nucleotide sequences of the downstream primers VH-R1 to VH-R10 of the heavy chain variable region are shown as SEQ ID NO:33 to SEQ ID NO:11, respectively. As shown in NO:42; the nucleotide sequences of the upstream primers VL-F1 to VL-F10 of the light chain variable region are shown in SEQ ID NO:43 to SEQ ID NO:52, respectively; the nucleotide sequences of the downstream primers VL-R1 to VL-R10 of the light chain variable region are shown in SEQ ID NO:53 to SEQ ID NO:62, respectively.

[0010] As a preferred embodiment, step 4, the SELEX screening includes: pre-cleaning with blank epoxy magnetic beads, followed by 10-18 rounds of forward screening, with the incubation time shortened, the number of washes increased, and the salt concentration increased in each round; from rounds 3-9 onwards, structural analogs are introduced for reverse screening; after each round of screening, ssDNA libraries are prepared by PCR amplification and λ exonuclease digestion; and finally, nucleic acid aptamers are obtained by sequencing and structural prediction; the nucleotide sequence of the nucleic acid aptamer is any one of SEQ ID NO:13 to SEQ ID NO:22.

[0011] As a preferred embodiment, the synthesis of the hapten in step 1 specifically includes the following steps: using 1-3 g of methyl 3-amino-5-ethoxybenzoate as a raw material, dissolving it in 12-36 mL of organic solvent, adding 1 mol / L-4 mol / L sodium hydroxide solution to adjust the pH to 5.0-8.0, extracting with ethyl acetate, and washing the extract phase three times each with water and petroleum ether, 10-30 mL each time, to obtain the purified 3-amino-5-ethoxybenzoic acid hapten.

[0012] As a preferred embodiment, the synthesis of the complete antigen in step 2 specifically includes the following steps: Step 2a, amino protection: Dissolve 3-8 g of 3-amino-5-ethoxybenzoic acid in 6-16 mL of tetrahydrofuran, add 2.37-6.32 mL of N,N-dimethylformamide and 2.19-5.84 mL of di-tert-butyl dicarbonate in an ice bath, and react at room temperature in the dark for 5-10 h; pour the reaction solution into 40-80 mL of ice water, adjust the pH to 3.0-4.0 with hydrochloric acid, and a solid precipitates; let stand at 4℃ for 1-2 h, filter, wash with ice water until neutral; purify by silica gel column chromatography with dichloromethane / methanol as eluent, collect the product and evaporate to dryness by rotary evaporation to obtain 3-(tert-butoxycarbonylamino)-5-ethoxybenzoic acid; Step 2b, adding the spacer arm: Take 200-600 mg of the product obtained in step 2a, dissolve it in 4-12 mL of anhydrous dichloromethane, and add 126.7-380.1 mg of the spacer arm, 160.7-482.1 mg of 1-hydroxybenzotriazole, and 227.9-683.7 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride sequentially with stirring in an ice bath. Remove the ice bath and react at room temperature in the dark for 10-18 h. After the reaction is complete, concentrate to remove dichloromethane, dissolve the residue in ethyl acetate or dichloromethane, wash successively with 0.1 mol / L dilute hydrochloric acid and water, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. Step 2c, coupling with the carrier protein: Using the carbodiimide method, take 12-25 mg of the product obtained in step 2b, dissolve it in 0.6-1.8 mL of N,N-dimethylformamide, add 24.75-51.9 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 22.4-46.7 mg of N-hydroxysuccinimide, and activate at room temperature for 2-4 h; separately, dissolve 28.8-81.4 mg of the carrier protein in 4-12 mL of phosphate buffer at pH 7.4, add the activation solution dropwise to the protein solution under ice bath, and react at 4°C in the dark for 12-24 h; the carrier protein is bovine serum albumin or ovalbumin; Step 2d, Deprotection of protecting group: After the coupling reaction is completed, 2.5-6.4 mL of trifluoroacetic acid is added dropwise to the reaction solution under ice bath conditions. The reaction is stirred under ice bath conditions for 1 h in the dark. Then, the pH is adjusted to 7.2-7.4 with 1 mol / L sodium hydroxide solution under ice bath conditions to terminate the reaction. Step 2e, purification: Transfer the reaction solution to a dialysis bag and dialyze it in 0.01 mol / L pH 7.4 phosphate buffer at 4°C for 24-72 h. After dialysis, transfer the solution in the bag to a centrifuge tube and centrifuge at 10000 r / min at 4°C for 10 min. Collect the supernatant to obtain the complete antigen.

[0013] As a preferred embodiment, the preparation method of the antibody-aptamer conjugate in step 5 is as follows: An amino-modified antibody is reacted with N-hydroxysuccinimide-biotin reagent at a molar ratio of 1:10-20 to obtain a biotinylated antibody; an amino-modified aptamer is reacted with N-hydroxysuccinimide-biotin reagent at a molar ratio of 1:10-25 to obtain a biotinylated aptamer; the biotinylated antibody is mixed with streptavidin at a molar ratio of 2-6:1 and incubated at room temperature to form a streptavidin-antibody complex; then, the biotinylated aptamer is added at a molar ratio of aptamer to streptavidin of 2-5:1, incubated at room temperature, and purified by gel filtration chromatography to obtain the final product.

[0014] As a preferred embodiment, the nano-sensitizing material in step 6 is any one of MXene / AuNPs, MXene / GO-rGO, or GO-rGO / perovskite; and the perovskite is any one of CsPbX3, MAPbX3, FAPbX3, or Cs2AgBiBr6.

[0015] Secondly, the present invention provides an electrochemical aptamer-antibody dual recognition sensor prepared by the above-described construction method.

[0016] Thirdly, the present invention provides the application of the above-mentioned sensor in detecting 3-aminophenethyl ether in animal-derived foods, comprising the following steps: Step 1, Sample pretreatment: Take animal food samples, mince or homogenize them, add extraction reagent for extraction, precipitate protein with sodium sulfate, centrifuge, collect the supernatant, dilute with phosphate buffer to obtain sample treatment solution; Step 2: Contact the working electrode of the electrochemical aptamer-antibody dual recognition sensor with the sample processing solution obtained in Step 1, and incubate it under constant temperature conditions to allow 3-aminophenethyl ether in the sample to bind to the recognition element on the electrode surface. Step 3: After incubation, clean the electrode and place it in potassium chloride test solution containing potassium ferrocyanide / potassium ferrocyanide. Use differential pulse voltammetry to detect the oxidation peak current value I. Step 4: Using the oxidation peak current value of the blank solution as I0, calculate the current change ΔI = I0 - I. Substitute ΔI into the standard curve showing a linear relationship between the concentration of 3-aminophenethyl ether and ΔI to calculate the content of 3-aminophenethyl ether in the animal food sample.

[0017] As a preferred embodiment, the animal-derived food in step 1 is any one of eggs, chicken meat, chicken liver, or chicken kidney; the extraction reagent is any one of methanol, acetonitrile, n-hexane, or a trichloroacetic acid-acetonitrile mixture, wherein the volume ratio of trichloroacetic acid to acetonitrile in the trichloroacetic acid-acetonitrile mixture is 1:5-10.

[0018] According to the above technical solution, the beneficial effects of the present invention are: 1. This invention combines biological antibodies with nucleic acid aptamers through rational design and controllable assembly to construct a dual-recognition sensing interface integrating aptamers and antibodies. It utilizes the spatial synergistic effect of the two recognition elements to enhance the capture ability and specificity of target molecules. At the same time, it introduces high-performance composite materials as the sensing substrate, which greatly improves electron transfer efficiency and forms abundant coupling sites, thereby fundamentally improving the selectivity, detection sensitivity and stability of the sensor for 3-aminophenethyl ether.

[0019] 2. This invention synergistically applies antibody and aptamer dual recognition elements to the same sensing interface and introduces nano-sensitizing materials (such as MXene / AuNPs, GO-rGO / perovskite, etc.) for signal amplification, significantly improving detection sensitivity. Research results show that the detection limit for 3-aminophenethyl ether is as low as 0.05 μg / kg, with a linear range of 0.5–400 μg / kg (r≥0.9999), far below the maximum residue limit stipulated by national regulations, meeting the requirements for trace detection.

[0020] 3. By employing dual recognition through both antibodies and aptamers, and utilizing the synergistic capture of different epitopes of the target molecule by the two recognition elements, cross-reactivity with structural analogs is effectively reduced. Experimental results show that the cross-reactivity rate of the antibody with six structural analogs, including o-aminophenethyl ether, p-aminophenethyl ether, and 3-aminobenzoic acid, is less than 1%, and the cross-reactivity rate of the aptamer with structural analogs is less than 1.8%, with a relative standard deviation of less than 5%, demonstrating good specificity, stability, and resistance to matrix interference.

[0021] 4. This invention uses an electrochemical detection platform, which does not require large-scale instruments and equipment. Sample pretreatment is simple, and the detection process can be completed in a short time. It is suitable for rapid on-site screening and overcomes the shortcomings of traditional instrument methods, such as expensive equipment, complex pretreatment, and insufficient specificity and poor stability of single recognition element sensors. Attached Figure Description

[0022] Figure 1 This is a synthetic route diagram of the hapten in an embodiment of the present invention.

[0023] Figure 2 The images shown are the nuclear magnetic resonance spectra of the hapten in this embodiment of the invention, where Figure A is the proton spectrum and Figure B is the carbon spectrum.

[0024] Figure 3 This is a synthetic route diagram of the complete antigen in an embodiment of the present invention.

[0025] Figure 4UV scan of the complete antigen; where AEBA is 3-amino-5-ethoxybenzoic acid, BSA is bovine serum albumin, OVA is oval serum albumin, AEBA-BSA is a conjugate of 3-amino-5-ethoxybenzoic acid and bovine serum albumin, and AEBA-OVA is a conjugate of 3-amino-5-ethoxybenzoic acid and oval serum albumin.

[0026] Figure 5 Complete antigen mass spectrum; where AEBA-BSA is a 3-amino-5-ethoxybenzoic acid-bovine serum albumin conjugate, and BSA is bovine serum albumin. Detailed Implementation

[0027] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0028] This invention provides a novel electrochemical biosensing method for detecting food hazards, based on 3-amino-5-ethoxybenzoic acid as a hapten, using a specific artificial antigen prepared by coupling it with a carrier protein, obtaining antibodies through animal immunization, and using this antigen-antibody recognition system as the core, combined with a specific aptamer, and electrodes modified with sensitizing materials to construct an electrochemical biosensing interface. The following sections provide a detailed description of each part.

[0029] Example 1

[0030] A method for constructing a 3-aminophenethyl ether dual-recognition electrochemical biosensor.

[0031] (1) Synthesis of haptens Synthetic routes such as Figure 1 As shown, 1 g of methyl 3-amino-5-ethoxybenzoate was placed in 12 mL of methanol solution at room temperature, and 1 mL of sodium hydroxide solution was added. The reaction system was stirred at room temperature for 12 h. A petroleum ether / ethyl acetate (3:1, v:v) was prepared as the developing solvent. Thin-layer chromatography (TLC) was used to check if the reaction was complete. The methanol was removed by concentration, and the mixture was diluted with ultrapure water. Concentrated hydrochloric acid was added dropwise, precipitating a large amount of solid. After stirring and standing, the mixture was filtered, and the resulting filter cake / filtrate was collected. The filter cake was washed three times each with 10 mL of water and petroleum ether, and the solid was collected and dried to obtain a pale yellow solid. The filtrate was extracted with ethyl acetate, and the extract phases were combined, concentrated, and dried to obtain the 3-amino-5-ethoxybenzoic acid hapten. The 1H and 1C NMR spectra of the hapten are shown below. Figure 2As shown, Figure A is the proton spectrum and Figure B is the carbon spectrum.

[0032] (2) Synthesis of complete antigen Amino protection: 3 g of 3-amino-5-ethoxybenzoic acid was dissolved in 6 mL of tetrahydrofuran. 2.37 mL of N,N-dimethylformamide and 2.19 mL of di-tert-butyl dicarbonate were added under ice bath conditions, and the reaction was carried out at room temperature in the dark for 5 h. The reaction solution was poured into 40 mL of ice water, and the pH was adjusted to 3.0 with hydrochloric acid, resulting in the precipitation of a solid. The solid was allowed to stand at 4 °C for 1 h, filtered, and washed with ice water until neutral. The crude product was purified by silica gel column chromatography using dichloromethane-methanol (8:1, v / v) as the eluent to give 3-(tert-butoxycarbonylamino)-5-ethoxybenzoic acid.

[0033] Adding the spacer arm: Dissolve 200 mg of 3-(tert-butoxycarbonylamino)-5-ethoxybenzoic acid in 4 mL of anhydrous dichloromethane. Stirring in an ice bath, add 126.7 mg of 6-aminohexanoic acid and 160.7 mg of 1-hydroxybenzotriazole sequentially, and finally add 227.9 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. Remove the ice bath and react at room temperature in the dark for 12 h. After the reaction is complete, concentrate the reaction solution to remove dichloromethane. Dissolve the residue in a small amount of ethyl acetate and transfer to a separatory funnel. Wash with 0.1 mol / L dilute hydrochloric acid to remove urea byproducts from the condensation of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and residual 1-hydroxybenzotriazole. Wash with water to remove residual acid and salt. Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the crude product.

[0034] Coupling with carrier proteins: Using the carbodiimide method, 12 mg of hapten was dissolved in 0.6 mL of anhydrous N,N-dimethylformamide, and 24.75 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 22.4 mg of N-hydroxysuccinimide were added. Activation was carried out at room temperature for 2 h. Separately, 39.4 mg of bovine serum albumin or 22.9 mg of ovalbumin was dissolved in 4 mL of 0.05 mol / L phosphate buffer (pH 7.4). The activation solution was added dropwise to the protein solution under ice bath conditions, and the reaction was carried out in the dark for 24 h.

[0035] Deprotection of the protecting group: After the coupling reaction was completed, 2.5 mL of trifluoroacetic acid was added dropwise to the reaction solution under ice bath conditions. The mixture was stirred in the ice bath in the dark for 1 h to complete the removal of the protecting group and expose the amino epitope of the hapten. After the reaction was completed, the pH of the system was slowly adjusted to 7.2 with 1 mol / L sodium hydroxide solution under ice bath conditions to terminate the reaction and obtain the coupling solution.

[0036] Purification: Transfer the above reaction solution to a dialysis bag and dialyze in 0.01 mol / L phosphate buffer (pH 7.4) at 4°C for 72 h. After dialysis, transfer the solution from the dialysis bag to a centrifuge tube, centrifuge at 10,000 r / min for 10 min at 4°C, collect the supernatant, which is the complete antigen, and store at -20°C.

[0037] The synthetic route of the complete antigen is as follows Figure 3 As shown.

[0038] Characterization of the complete antigen: The prepared complete antigen and carrier protein were subjected to ultraviolet spectroscopy scans, and the positions of their maximum absorption peaks were compared. Figure 4 The molecular weight of the complete antigen was determined by time-of-flight mass spectrometry, confirming the covalent binding of the hapten to the carrier protein. The coupling ratio of the 3-amino-5-ethoxybenzoic acid hapten to bovine serum albumin was identified as 18.8:1, and the coupling ratio to ovalbumin was 14.9:1. Figure 5 The ultraviolet spectroscopy showed that the maximum absorption peak of the complete antigen changed significantly compared to the hapten and carrier protein, proving that the complete antigen was successfully prepared.

[0039] (3) Antibody preparation Polyclonal antibodies (for monitoring immunization efficacy): Using a hapten-protein conjugate as the immunogen, a 1 mg / mL immunogen solution was mixed with an equal volume of graphene oxide dispersion (0.5 mg / mL), and then thoroughly vortexed with Freund's adjuvant at a 1:1 volume ratio to form a stable water-in-oil emulsion. Female BALB / c mice aged 6-8 weeks were selected as immunization animals, and immunization was performed via subcutaneous injection at multiple sites on the back, with each mouse receiving 100 mL per injection. The initial immunization used Freund's complete adjuvant, followed by booster immunizations every two weeks using Freund's incomplete adjuvant, for a total of three booster immunizations. Seven days after the final immunization, a small amount of blood was collected from the tail, and antibody titers were determined using an indirect competitive ELISA method. After the collected anticoagulated blood was allowed to stand at 4°C for stratification, the supernatant serum was collected, aliquoted, and stored at -20°C.

[0040] Preparation of monoclonal antibodies: Spleens from qualified BALB / c mice (6-8 weeks old, female) were aseptically ground and filtered to prepare a spleen cell suspension. Simultaneously, SP2 / 0 myeloma cells in logarithmic growth phase (approximately 1 × 10⁻⁶ cells) were resuscitated and cultured. 7Spleen cells were mixed with SP2 / 0 cells at a ratio of 6:1, and the supernatant was discarded after centrifugation. In a 37°C water bath, 1 mL of 50% polyethylene glycol was slowly added as a fusion agent and gently mixed. The fusion reaction was then terminated by slowly adding preheated serum-free Durbeco modified Eagle medium. The fused cells were resuspended in selective complete medium containing 1× hypoxanthine-aminopterin-thymidine medium, and the cell density was adjusted to 1×10⁶ cells / mL. 5 Cells were seeded at a density of 100 μL / mL into 96-well cell culture plates. The plates were incubated at 37°C in a 5% CO2 incubator. After 7-14 days of culture, when visible cell clones appeared in the wells, the culture supernatant was aspirated, and the 3-aminophenethyl ether specific antibody was detected using an indirect ELISA method. Strongly positive wells (OD200) were selected. 450 (If the value is more than 2.1 times higher than the negative control), subcloning is performed using the limiting dilution method: after diluting the cells, they are seeded into new 96-well plates (e.g., 0.5-1 cells per well) and cultured. This process is repeated 2-3 times until a monoclonal hybridoma cell line that can stably secrete antibodies is obtained. The identified monoclonal cell line is expanded and cultured. A portion is used to prepare ascites fluid or to collect the supernatant from in vitro culture to obtain antibodies; the other portion is added to cryopreservation solution (fetal bovine serum containing 10% dimethyl sulfoxide) and stored in liquid nitrogen for long-term preservation. BALB / c mice that have been pre-injected with 0.5 mL of norperidine are injected intraperitoneally with 5 × 10⁵ mmol / L of phenazine. 6 Hybridoma cells were collected. Ascites fluid was collected after the mouse abdomen became significantly distended. The ascites fluid or cell culture supernatant was centrifuged to remove cell debris, then purified using a protein A / G affinity chromatography column. The purified antibody samples were aliquoted and stored at -20°C.

[0041] (4) Antibody performance evaluation and characterization The titer of the monoclonal antibodies in the three mice reached the expected level (see Table 1), indicating that the artificial antigen was successfully synthesized and had good immunogenicity. After five immunizations, the target substance showed a significant inhibitory effect on the antibody, laying the foundation for subsequent applications.

[0042] Table 1. Determination of serum titer of 3-aminophenethyl ether murine monoclonal antibody

[0043] The results of the specificity study of the monoclonal antibody showed that the cross-reactivity rate of the antibody with the major structural analogues of 3-aminophenethyl ether (o-aminophenethyl ether, p-aminophenethyl ether, 3-aminobenzoic acid, 3-aminoanisole, 3-amino-5-ethoxybenzoic acid, 3-ethoxybenzoic acid) was less than 1%, indicating no significant cross-reactivity and good specificity. 50With a concentration of 14.63 ng / mL, the antibody exhibits high sensitivity, meeting the requirements for trace detection. Precision evaluation results show an intra-assay coefficient of variation of less than 4% and an inter-assay coefficient of variation of less than 5%, indicating good repeatability and meeting precision standards for the antibody detection system. In the practical sample suitability evaluation, the sample spiked recoveries ranged from 81.36% to 102.43%, with relative standard deviations ranging from 0.84% ​​to 2.86%, demonstrating stable antibody binding in actual sample matrices and strong anti-interference properties, making it suitable for practical sample detection. Affinity assay results show that the antibody has a strong binding affinity to 3-aminophenethyl ether, enabling efficient recognition of the target molecule and meeting the needs of subsequent immunoassay applications.

[0044] Monoclonal antibody variable region sequence analysis includes five steps: total RNA extraction from hybridoma cells, reverse transcription to synthesize cDNA, PCR amplification of the antibody variable region gene, cloning and sequencing of the PCR product, and nucleotide sequencing and amino acid sequence analysis. The specific operation is as follows.

[0045] ① Total RNA extraction from hybridoma cells: Cells cultured to the logarithmic growth phase (approximately 1×10⁻⁶) will be subjected to total RNA extraction. 6 Up to 5×10 6 Mouse hybridoma cells (approximately 80%-90% density) were transferred into sterile centrifuge tubes and centrifuged at 1000 r / min for 4 hours. o Centrifuge for 5 min, discard the supernatant, add 5 mL of pre-chilled PBS buffer, gently resuspend the cells by pipetting, repeat centrifugation and washing twice to thoroughly remove culture medium residue, and finally retain approximately 1×10⁶ cells. 6 Prepare at least one cell pellet for later use. Handle the cells gently during the procedure to avoid damaging them and affecting subsequent RNA extraction.

[0046] Add 1 mL of Trizol reagent to the cell pellet, vortex vigorously for 30 s to fully lyse the cells, incubate at room temperature for 5 min, then add 0.2 mL of chloroform, shake vigorously for 15 s, and incubate at room temperature for 2-3 min to allow the solution to separate into layers. Centrifuge at 12000 r / min, 4℃ for 15 min, carefully aspirate the upper aqueous phase into a new RNase-free EP tube, avoiding the middle protein phase. Add an equal volume of isopropanol and mix inverted. Incubate at room temperature for 10 min to allow the RNA to fully precipitate. Centrifuge again and discard the supernatant. Wash the RNA pellet with 1 mL of 75% RNase-free ethanol, centrifuge again and discard the supernatant, then air dry at room temperature for 5-10 min (avoid complete drying). Dissolve the RNA in 20-50 μL of RNase-free deionized water and store at -80℃ for later use. Simultaneously, take 2 μL of RNA for agarose gel electrophoresis and nucleic acid concentration determination to verify RNA integrity and purity, and ensure the OD of the obtained RNA. 260 / OD 280The ratio was between 1.8 and 2.0, and the 28 S and 18 S ribosomal RNA bands were clearly visible and in a normal ratio (approximately 2:1) as confirmed by agarose gel electrophoresis.

[0047] ② Reverse transcription to synthesize cDNA: Using extracted total RNA as a template, cDNA is synthesized using reverse transcriptase. Oligo(dT) primers and / or random primers are used during reverse transcription to ensure coverage of the complete mRNA sequence, especially including the coding region of the antibody variable region. A negative control without reverse transcriptase should be included in the reaction system to rule out the possibility of genomic DNA contamination. Prepare a reverse transcription reaction solution in an RNase-free EP tube with a total volume of 20 μL. Add 5 μg total RNA, 1 μL Oligo(dT) primer, 2 μL dNTP mixture, 0.5 μL RNase inhibitor, and an appropriate amount of RNase-free deionized water to a final volume of 14 μL. After mixing, incubate at 65 °C for 5 min and then immediately on ice for 5 min to denature the RNA and allow the primers to bind. Add 4 μL reverse transcriptase buffer and 1 μL reverse transcriptase, mix well, and incubate at 42 °C for 60 min for reverse transcription. Inactivate the reverse transcriptase by incubating at 70 °C for 15 min. After the reaction, store the cDNA solution at -20 °C for later use to avoid repeated freeze-thaw cycles that could affect the quality of the cDNA.

[0048] ③ PCR amplification of antibody variable region genes: Specific amplification of the heavy chain variable region (VH) and light chain variable region (VL, including κ and λ types) genes of antibodies was performed. Specific primers were designed based on conserved regions of the mouse antibody genes, and nested PCR or falling PCR strategies were employed to improve the specificity and efficiency of amplification. Primer design primarily targeted the IGHV family signal peptide sequence and the 5' end of IGHG, as well as the IGKV / IGLV signal peptide sequence and the 5' end of IGKC / IGLC.

[0049] The specific primer sequences for the variable region of the mouse antibody used in this invention are as follows: Heavy chain variable region (VH) upstream primers (SEQ ID NO:23 to SEQ ID NO:32): VH-F1: GAGGTGCAGCTGCAGGAGTCTGG VH-F2: GAGGTTCAGCTGCAGCAGTCT VH-F3: CAGGTGCAGCTGGTGCAGTCTGG VH-F4:CAGGTCCAGCTGCAGGAGTCGGG VH-F5: GAAGTGCAGCTGGTGGAGTCTGG VH-F6: GAGGTGAACTGGTGGAGTCTGG VH-F7: GATGTGAAGTTCCCAGAGTCTG VH-F8: ATGGACTGGAGCTGGGTCTTC VH-F9: ATGAAATGCAGCTGGGTC VH-F10: GAGGTGCAGCTTCAGGAGTCGGG Heavy chain variable region (VH) downstream primers (SEQ ID NO:33 to SEQ ID NO:42): VH-R1: TGAGGAGACGGTGACCAGGGT VH-R2: TGAGGAGACGGTGACTGAGGTTC VH-R3: CTGAGGAGACGGTGACCGTGG VH-R4: TGACCAGAGTGGATAGACGGTGACCG VH-R5: GGTTGACCAGAGTGGATAGACG VH-R6: GGAAGGTGTGCACACCGCTGGAC VH-R7: GGAAGGTGTGCACACTGCTGGAC VH-R8: ACTCAGGGAAGTAGCCCTTGAC VH-R9: GCTGCTGAGGAGACGGTGAC VH-R10: TGAGGAGACGGTGACTGAGGTTCC Light chain variable region (VL) upstream primers (SEQ ID NO:43 to SEQ ID NO:52): VL-F1: GACATCCAGATGACCCAGTCTCC VL-F2: GACATTGTGATGACCCAGTCTCC VL-F3: GAAATTGTGCTGACCCAGTCTCC VL-F4: GATATCCAGATGACCCAGTCTCC VL-F5: GATGTGCTCACCCAGTCTCC VL-F6: CAGTCTGTGCTGACCCAGACTGA VL-F7:GACATTGTGATGACCCAGACTCC VL-F8:GACATCCAGCTGACCCAGTCTCC VL-F9: GATGTGATGACCCAGTCTCCA VL-F10: GAAACAGCTCTTCCGTGACCCAG Light chain variable region (VL) downstream primers (SEQ ID NO:53 to SEQ ID NO:62): VL-R1: TGATTTCCAGCTTGGTCCC VL-R2: TTTGATTTCCAGCTTGGTCCC VL-R3: AGCCCGTTTGATTTCCAGCTTGGTG VL-R4:TTTGATTTCCAGCTTGGTCCCCTCC VL-R5: GTCCTGGACGGTCAGCTTGGT VL-R6: CTAGGACGGTCAGCTTGGTCCC VL-R7: CCGTTTTATTTCCAGCTTGGTCCC VL-R8: GAGTTGGGTGACAGCTGG VL-R9: GACAGGCTGGAGGGGTACC VL-R10:GGATACAGTTGGTGCAGCATC In this embodiment, the primer combinations actually used were: mouse IgG VH upstream primers (VH-F1, VH-F4, VH-F7) and VH downstream primers (VH-R3, VH-R5, VH-R9); mouse IgG VL upstream primers (VL-F4, VL-F7, VL-F8) and VL downstream primers (VL-R2, VL-R4, VL-R5). All primers were at a concentration of 10 μmol / L and could be directly used for PCR amplification. PCR reaction systems for the VH and VL genes were prepared separately (total system 50 μL): 2 μL cDNA template, 2 μL upstream primers, 2 μL downstream primers, 0.5 μL (2.5 U) Taq DNA polymerase, 5 μL 10× PCR buffer, and Mg... 2+Add 3 μL of dNTP mixture and 2 μL of dNTP mixture (2.5 mmol / L each), and bring the total volume to 50 μL with sterile deionized water. Mix well and briefly centrifuge to concentrate the reaction solution at the bottom of the tube. PCR program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 45 s, for 35 cycles; 72℃ final extension for 10 min, and incubate at 4℃. After the PCR reaction, take 10 μL of product for 1.5% agarose gel electrophoresis and recover the PCR product using a DNA gel recovery kit.

[0050] ④PCR product purification and cloning: The specifically amplified PCR band (approximately 350-400 bp) was excised from the agarose gel and purified using a gel extraction kit. The purified DNA product was ligated into a TA cloning vector (such as pMD18-T or pGEM-T), transformed into competent E. coli cells, and single colonies were selected by blue-white screening. Plasmids were extracted and validated. The specific steps are as follows. The recovered PCR products were ligated with the TA vector (total system 10 μL: 1 μL T vector, 4 μL recovered PCR product, 1 μL ligase buffer, 1 μL T4 DNA ligase, 3 μL sterile water). After mixing, the mixture was incubated overnight at 16 ℃ to ensure complete ligation. The ligation product was transformed into competent E. coli cells, incubated on ice for 30 min, then heat-shocked at 42 ℃ for 90 s, followed by an immediate ice incubation for 2 min. 450 μL of antibiotic-free LB medium was added, and the cells were thawed at 37 ℃ and 200 r / min for 1 h. 100 μL of the culture was spread on LB solid medium containing ampicillin (final concentration 100 μg / mL) and incubated upside down overnight at 37 ℃. A single colony was picked and inoculated into LB liquid medium containing ampicillin and cultured with shaking for 8-10 h. 1 μL of the bacterial culture was used as a template for PCR primer verification. Colonies that detected the target band by electrophoresis were considered positive clones.

[0051] ⑤ Nucleotide Sequencing and Amino Acid Sequencing: Verified positive clone plasmids are sent for sequencing using universal primers (e.g., M13 Forward / Reverse) for bidirectional sequencing. The obtained nucleotide sequences are analyzed using bioinformatics software (e.g., SnapGene, Geneious, or online tools such as IMGT / V-QUEST). By comparing with mouse antibody gene databases, the origin of the gene fragment is determined, and the nucleotide sequence is translated into an amino acid sequence using the genetic code table to obtain the complete amino acid sequence of the antibody variable region. Specifically, the sequencing primers are M13F (5'-TGTAAAACGAC GGCCAGT-3') and M13R (5'-CAGGAAACAGCTATGACC-3'). At least three positive clones are selected for sequencing for each fragment to ensure sequence accuracy. The sequencing results are then processed to obtain the complete nucleic acid sequences of VH and VL, which are then translated into amino acid sequences. Using databases such as IMGT and IgBLAST, the frame regions (FR1-FR4) and complementarity-determining regions (CDR1-CDR3) in the amino acid sequences were annotated to determine the structure of the antibody variable region. Simultaneously, the sequencing results of three clones were compared to verify the consistency of the VH and VL sequences, eliminating sequencing errors and mutations. The amino acid and nucleotide sequences of the heavy and light chain variable regions of the obtained monoclonal antibody are as follows. The amino acid sequence of the heavy chain variable region of the obtained monoclonal antibody sequence 1 is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:2. The amino acid sequence of the light chain variable region is shown in SEQ ID NO:3, and the nucleotide sequence is shown in SEQ ID NO:4.

[0052] Monoclonal antibody sequence 1, heavy chain variable region amino acids (SEQ ID NO:1) MKSLSLLVLSATGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWLEWVKQRPGQGLEWIGEINHDGGGTNYNEKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARRGYYGSSYFFDVWGAGTTVTVSS Monoclonal antibody sequence 1 heavy chain variable region nucleotides (SEQ ID NO:2) ATGAAGAGCCTGAGCCTGCTGGTGCTGAGCGCGACCGGCGTGCATAGCCAGGTGCAGCTGCAGCAGCCGGGCGCGGAACTGGTGAAACCGGGCGCGAGCGTGAAACTGAGCTGCAAAGCGAGCGGCTATACCTTTACCAGCTATTGGCTGGAATGGGTGAAACAGCGCCCGGGCCAGGGCCTGGAATGGATTGGCGAAATTAACCATGATGGCGGCGGCACCAACTATAACGAAAAATTTAAAGGCAAAGCGACCCTGACCGTGGATAAAAGCAGCAGCACCGCGTATATGCAGCTGAGCAGCCTGACCAGCGAAGATAGCGCGGTGTATTATTGCGCGCGCCGCGGCTATTATGGCAGCAGCTATTTTTTTGATGTGTGGGGCGCGGGCACCACCGTGACCGTGAGCAGC Amino acid sequence of monoclonal antibody sequence 1 light chain variable region (SEQ ID NO:3) DIVMTQSPSSLAVSAGEKVTMSCRASHNIGHSLAWYQQKPGQSPKLLIYRASKLKRGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQYKRHPRSFGGGTKLEIK Nucleotide sequence of monoclonal antibody sequence 1 light chain variable region (SEQ ID NO:4) GATATTGTGATGACCCAGAGCCCGAGCAGCCTGGCGGTGAGCGCGGGCGAAAAAGTGACCATGAGCTGCCGCGCGAGCCATAACATTGGCCATAGCCTGGCGTGGTATCAGCAGAAACCGGGCCAGAGCCCGAAACTGCTGATTTATCGCGCGAGCAAACTGAAACGCGGCGTGCCGGATCGCTTTACCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCGTGCAGGCGGAAGATCTGGCGGTGTATTATTGCCAGCAGTATAAACGCCATCCGCGCAGCTTTGGCGGCGGCACCAAACTGGAAATTAAA The heavy and light chain gene sequences were compared and analyzed in the NCBI database. The results showed that the heavy chain variable region gene sequence of the prepared 3-aminophenethyl ether monoclonal antibody showed the highest homology with the mouse immunoglobulin heavy chain variable region gene sequence (GenBank: MN700932.1), with 274 / 295 matching bases and a homology of 93%. Its heavy chain variable region amino acid sequence showed the highest homology with the mouse immunoglobulin heavy chain variable region amino acid sequence (Sequence-ID: AAA67438.1), with 112 / 132 matching amino acids and a homology of 85%. Regarding the light chain variable region, its gene sequence showed the highest homology with the mouse immunoglobulin light chain variable region gene sequence (GenBank: KY038040.1), with 177 / 207 matching bases and a homology of 86%. The amino acid sequence of its light chain variable region showed the highest homology with the amino acid sequence of the mouse immunoglobulin light chain variable region (Sequence-ID: 7UPD_E), with a matching amino acid count of 87 / 113 and a homology of 77%. Based on the above homology analysis of the heavy and light chain variable region genes and amino acid sequences, no sequences completely identical to those of this invention were found.

[0053] The CDR regions of the heavy chain and light chain variable region sequences were analyzed using the Kabat definition scheme at https: / / www.novopro.cn / tools / cdr.html.

[0054] Monoclonal antibody sequence 1: Antibody heavy chain CDR region: CDR-H1: SYWLE CDR-H2: EINHDGGGTNYNEKFKG CDR-H3: RGYYGSSYFFDV Antibody light chain CDR region: CDR-L1: RASHNIGHSLA CDR-L2: RASKLKR CDR-L3: QQYKRHPRS Using the same method, monoclonal antibody sequence 2 and monoclonal antibody sequence 3 were obtained from two other hybridoma cell lines, respectively. The amino acid sequence of the heavy chain variable region of monoclonal antibody sequence 2 is shown in SEQ ID NO:5, the nucleotide sequence is shown in SEQ ID NO:6, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7, and the nucleotide sequence is shown in SEQ ID NO:8; the amino acid sequence of the heavy chain variable region of monoclonal antibody sequence 3 is shown in SEQ ID NO:9, the nucleotide sequence is shown in SEQ ID NO:10, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:11, and the nucleotide sequence is shown in SEQ ID NO:12.

[0055] Monoclonal antibody sequence, SEQ ID NO:5, heavy chain variable region amino acid sequence. MGWSCIILFLVATATGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPNNGRTNYNEKFKNKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTRSYYGSSYWYFDVWGAGTTVTVSS Monoclonal antibody sequence, nucleotide sequence of the variable region of the heavy chain (SEQ ID NO:6) ATGGGCTGGAGCTGCATTATTCTGTTTCTGGTGGCGACCGCGACCGGCGTGCATAGCCAGGTGCAGCTGCAGCAGCCGGGCGCGGAACTGGTGAAACCGGGCGCGAGCGTGAAACTGAGCTGCAAAGCGAGCGGCTATAACCTTTACCAGCTATTGGATGCATTGGGTGAAACAGCGCCCGGGCCAGGGCCTGGAATGGATTGGCGAAAATT AACCCGAACAACGGCCGCACCAACTATAACGAAAAATTTAAAAACAAAGCGACCCTGACCGTGGATAAAAGCAGCAGCACCGCGTATATGCAGCTGAGCAGCCTGACCAGCGAAGATAGCGCGGTGTATTATTGCACCCGCAGCTATTATGGCAGCAGCTATTGGTATTTTGATGTGTGGGGCGCGGGCACCACCGTGACCGTGAGCAGC Monoclonal antibody sequence 2, light chain variable region amino acid sequence (SEQ ID NO:7) DIVMTQSPSSLAVSAGEKVTMSCRSSHPVHNDMHWYQQKPGQSPKLLIYMSRSAERGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCMHFSNRRSTRMYTFGGGTKLEIK Monoclonal antibody sequence 2, light chain variable region nucleotide sequence (SEQ ID NO:8) GATATTGTGATGACCCAGAGCCCGAGCAGCCTGGCGGTGAGCGCGGGCGAAAAAGTGACCATGAGCTGCCGCAGCAGCCATCCGGTGCATAACGATATGCATTGGTATCAGCAGAAACCGGGCCAGAGCCCGAAACTGCTGATTTATATGAGCCGCAGCGCGGAAC GCGGCGTGCCGGATCGCTTTACCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCGTGCAGGCGGAAGATCTGGCGGTGTATTATTGCATGCATTTTAGCAACCGCCGCAGCACCCGCATGTATACCTTTGGCGGCGGCACCAAACTGGAAATTAAA The CDR region of monoclonal antibody sequence 2 was analyzed using the same method, and the results are as follows: Monoclonal antibody sequence 2: Heavy chain variable region CDR region CDR-H1: SYWMH CDR-H2: EINPNNGRTNYNEKFKN CDR-H3: SYYGSSYWYFDV Light chain variable region CDR region CDR-L1: RSSHPVHNDMH CDR-L2: MSRSAER CDR-L3: MHFSNRRSTRMYT Monoclonal antibody sequence, amino acid sequence of the variable region of the heavy chain (SEQ ID NO:9) MKYLSILFLVATGVTSQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWIHWVKQRPGQGLEWIGEINPNAGGTNYNEKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARRGYYGSSYWYFDVWGAGTTVTVSS Monoclonal antibody sequence, nucleotide sequence of the variable region of the triplet chain (SEQ ID NO:10) ATGAAGTACCTGTCCATCCTGTTCTTGGTGGCAACAGGCGTCCACTCCCAGGTGCAGCTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATCCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTGGAGTGGATCGGCGAGATTAATCCT AACGCTGGTGGTACTAACTACAACGAGAAGTTCAAGGGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAAGACTCTGCGGTCTATTACTGTGCAAGACGCGGCTACTACGGTAGTAGCTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA The amino acid sequence of the light chain variable region of the monoclonal antibody sequence 3 (SEQ ID NO:11) DIVMTQSPSSLAVSAGEKVTMSCRASEDVDDEVNWYQQKPGQSPKLLIYDGNSRASGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCLQHHSFPRSFGGGTKLEIK Monoclonal antibody sequence 3 light chain variable region nucleotide sequence (SEQ ID NO:12) GACATCGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCTGCAGGCGAGAAGGTCACTATGAGCTGCCGGGCCAGCGAGGACGTGGACGACGAAGTGAACTGGTATCAGCAGAAGCCAGGGCAGTCTCCTAAGCTGCTGATCTACGACGGCAACAGCC GGGCCTCCGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTACTACTGCCTGCAGCACCACTCCTTCCCCAGGTCCTTCGGCGGAGGGACCAAGCTGGAGATCAAA The CDR region of monoclonal antibody sequence 3 was analyzed using the same method, and the results are as follows: Monoclonal antibody sequence 3: Heavy chain variable region CDR CDR-H1: SYWIH CDR-H2: EINPNAGGTNYNEKFKG CDR-H3: RGYYGSSYWYFDV Light chain variable region CDR CDR-L1: RASEDVDDEVN CDR-L2: DGNSRAS CDR-L3: LQHHSFPRS (5) Screening and evaluation of aptamers Using 3-aminophenethyl ether as the target, epoxy-based magnetic beads were employed to covalently immobilize the target molecule through a ring-opening reaction between the amino groups on the target molecule and the epoxy groups on the surface of the magnetic beads. An ssDNA library containing 40 random bases was used as the initial library for SELEX screening. During screening, the initial library was first incubated with blank epoxy-based magnetic beads at room temperature for 30 min for pre-cleaning to remove non-specifically binding sequences. Subsequently, 14 rounds of forward screening were performed: the first round of incubation was 60 min, and the incubation time was gradually shortened from the second round onwards, reaching 10 min by the tenth round; simultaneously, the number of high-salt washes was increased from 2 to 6, and the salt concentration was gradually increased from 0.1 mol / L to 0.6 mol / L to progressively improve the specificity of the screening. From the fifth round onwards, reverse screening was introduced: the library was incubated with magnetic beads immobilized with structural analogs for 50 min each at room temperature, and unbound libraries were collected for subsequent forward screening to remove sequences that non-specifically bind to the structural analogs. After each round of screening, the products were subjected to PCR amplification. The amplified products were then digested with λ exonuclease (37℃, 60 min) to prepare ssDNA libraries. After the libraries were enriched and stabilized, the final round of libraries were subjected to high-throughput sequencing. Candidate sequences were screened based on sequence and secondary structure prediction to obtain nucleic acid aptamers that specifically recognize 3-aminophenethyl ether. The sequence was sequenced, and its nucleotide sequence is shown in SEQ ID NO:13, AGCACAGAGGTCAGATGTAGACATCTGTGCTCTTCGCGGATCCGAGCTCGGTACCC (SEQ ID NO:13).

[0056] The binding performance of the screened aptamers was evaluated, and the dissociation constant between the aptamer and 3-aminophenethyl ether was 8.5 × 10⁻⁶. -8 It exhibits excellent affinity and strong, stable binding at mol / L; the cross-reactivity with structural analogs is less than 1.8%, demonstrating outstanding specificity. Within the temperature range of 25℃-37℃ and pH 5.0-8.5, the binding activity retention rate exceeds 94%, and its recognition ability is stable in real sample matrices, with a spiked recovery rate exceeding 89%, meeting the requirements for highly sensitive detection.

[0057] (6) Antibody-aptamer conjugation Antibody-aptamer conjugation was performed using a biotin-streptavidin directional immobilization method. 2 μL of the monoclonal antibody shown in Sequence 1 was dissolved in 500 μL of phosphate buffer. N-hydroxysuccinimide-biotin (dissolved in dimethyl sulfoxide, 2% concentration) was added at a monoclonal antibody to biotin molar ratio of 1:20. After incubation at room temperature for 1.5 h, dialyzing was performed to obtain the biotinylated antibody.

[0058] The aptamer was modified at the 5' end using the carbodiimide method to obtain the amino-modified aptamer. Separately, 8 nmol of the amino-modified aptamer was dissolved in 300 μL of 0.1 mol / L phosphate buffer (pH 8.0) and mixed with N-hydroxysuccinimide-biotin at a molar ratio of 1:25. After incubation at room temperature for 4 h, the mixture was purified by ultrafiltration to obtain the biotinylated aptamer. The biotinylated antibody was mixed with streptavidin at a molar ratio of 4:1 and incubated at room temperature for 45 min to form a streptavidin-antibody complex. Then, the biotinylated aptamer was added at a ratio of aptamer to initial streptavidin of 3:1 and incubated at room temperature for 45 min. The reaction solution was purified by gel filtration chromatography to obtain the target antibody-aptamer conjugate.

[0059] (7) Construction of dual-recognition electrochemical sensing interface The gold electrode was pretreated as follows: after polishing with 0.05 μm alumina powder, it was ultrasonically cleaned in ultrapure water and -0.2 V anhydrous ethanol for 3 min in sequence, and then activated by cyclic voltammetry in 0.5 mol / L sulfuric acid solution (scan range -1.6, scan rate 100 mV / s). After rinsing, it was dried with nitrogen gas to obtain the activated electrode.

[0060] Sensitizing material was modified onto the surface of the activation electrode: 10 μL of MXene / AuNPs composite dispersion with a concentration of 1 mg / mL was drop-coated onto the surface of the activation electrode, dried at room temperature for 2 h, rinsed 3 times with phosphate buffer (pH 7.4), and dried with nitrogen to construct the sensing interface.

[0061] Take 12 μL of 0.5% bovine serum albumin solution prepared with phosphate buffer and drop it onto the bottom surface. Incubate at room temperature for 1.5 h, rinse 3 times with phosphate buffer (pH 7.4), blow dry with nitrogen, and block non-specific binding sites.

[0062] Dilute the antibody-aptamer conjugate to 0.1 mg / mL with phosphate buffer, drop 12 μL onto the blocked substrate surface, incubate at room temperature for 3 h, rinse and dry, add 10 μL of 1% bovine serum albumin solution, block again at room temperature for 30 min, rinse and dry.

[0063] The electrode was placed in a potassium ferricyanide / potassium ferrocyanide solution and scanned using cyclic voltammetry (scan range -0.2-0.6V, scan rate 40 mV / s) to obtain the final dual-recognition working electrode, labeled BSA / Ab / Apt / Mxene / AμNPs / AμE.

[0064] (8) Electrochemical characterization Electrochemical characterization of each step in the electrode modification process was performed using cyclic voltammetry. The bare gold electrode and the layer-by-layer modified electrode were placed in a 0.1 mol / L potassium chloride test solution containing 5 mmol / L potassium ferrocyanide / potassium ferrocyanide, and the electrochemical responses were recorded. The CV scan range was -0.2–0.6 V, and the scan rate was 50 mV / s. The DPV parameters were set as follows: scan range -0.2–0.6 V, pulse amplitude 50 mV, pulse width 50 ms, and scan rate 50 mV / s. Experimental results showed that the materials were progressively modified onto the electrode surface, and the oxidation peak current exhibited a regular change compared to the bare gold electrode. This indicates that the modified materials were successfully immobilized on the electrode surface and possessed good electrochemical activity, providing an effective sensing interface for subsequent biorecognition.

[0065] (9) Performance analysis of electrochemical biosensors To evaluate the detection performance of the constructed electrochemical sensor for 3-aminophenethyl ether, differential pulse voltammetry was used to detect 3-aminophenethyl ether standards at different concentrations. 10 μL of 3-aminophenethyl ether standard solutions of different concentrations were dropped onto the surface of the modified electrode and incubated at 37℃ for 15 min to allow for sufficient binding of the antigen to the electrode surface. After incubation, the electrode was rinsed with 0.1 mol / L phosphate buffer (pH 7.4). Subsequently, the electrode was placed in a 0.1 mol / L potassium chloride test solution containing 5 mmol / L potassium ferrocyanide / potassium ferrocyanide, and the oxidation peak current value (I) corresponding to each concentration was recorded. The current change ΔI = I0 - I was calculated using the current value of the blank solution as I0, and a calibration curve was plotted with ΔI as the ordinate. Within the range of 0.5–400 μg / kg, the concentration of 3-aminophenethyl ether showed a linear relationship with ΔI, with a linear regression equation of Y = 1.0254 + 0.0074X, a correlation coefficient r of 0.9999, and a detection limit of 0.05 μg / kg. These results indicate that the constructed sensor possesses good sensitivity and a low detection limit, making it suitable for the detection of trace amounts of 3-aminophenethyl ether.

[0066] (10) Determination of the recovery rate of spiked chicken samples Pretreatment of chicken samples: Take 5 g of chicken sample, mince it, and transfer it to a 50 mL centrifuge tube. Add 5 g of sodium sulfate and 20 mL of methanol, vortex and mix for 15 min to precipitate proteins, then centrifuge at 10000 r / min for 10 min. Collect the supernatant and store it at 4℃. Before detection, dilute the supernatant with 0.01 mol / L phosphate buffer, and then use the prepared dual-recognition electrochemical biosensor to detect the diluted sample solution. The recoveries and relative standard deviations of different spike concentrations in chicken are shown in Table 2. The sample spike recoveries ranged from 87.29% to 91.40%, and the relative standard deviations ranged from 0.90% to 4.81%.

[0067] Table 2 Determination of spiked recovery rate of chicken samples

[0068] Example 2

[0069] The difference between this embodiment and Embodiment 1 is that: (1) Synthesis of haptens 2 g of methyl 3-amino-5-ethoxybenzoate was placed in 24 mL of methanol solution, and sodium hydroxide solution was added. The mixture was stirred at room temperature for 16 h. After the reaction was complete as detected by TLC, the methanol was removed by concentration, the solution was diluted with ultrapure water, and concentrated hydrochloric acid was added dropwise to precipitate a solid. The solid was then filtered. The filter cake was washed three times each with water and petroleum ether (20 mL each time), and the solid was collected and dried. The filtrate was extracted with ethyl acetate, and the extracts were combined and concentrated to obtain a solid, which was then dried. The two solid fractions were combined to obtain the 3-amino-5-ethoxybenzoic acid hapten. The rest was the same as in Example 1.

[0070] (2) Synthesis of complete antigens: Amino protection: Dissolve 5 g of 3-amino-5-ethoxybenzoic acid in 6 mL of tetrahydrofuran. Add 3.95 mL of N,N-dimethylformamide and 3.65 mL of di-tert-butyl dicarbonate in an ice bath and incubate at room temperature in the dark for 8 h. Pour the reaction solution into 60 mL of ice water and adjust the pH to 3.0-4.0 with hydrochloric acid, precipitating a solid. Let stand at 4 °C for 1 h, filter, and wash with ice water until neutral. Purify the crude product by silica gel column chromatography using dichloromethane-methanol (12:1, v / v) as eluent to give 3-(tert-butoxycarbonylamino)-5-ethoxybenzoic acid.

[0071] Adding the spacer arm: Dissolve 400 mg of 3-(tert-butoxycarbonylamino)-5-ethoxybenzoic acid in 5 mL of anhydrous dichloromethane. Under ice bath conditions, add 253.4 mg of 6-aminohexanoic acid, 321.4 mg of 1-hydroxybenzotriazole, and 455.8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride sequentially. React at room temperature in the dark for 15 h. The rest is the same as in Example 1.

[0072] Coupling with carrier proteins: Using the carbodiimide method, 16 mg of the above hapten was dissolved in 1.2 mL of anhydrous N,N-dimethylformamide, and 31.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 25.1 mg of N-hydroxysuccinimide were added. The mixture was activated at room temperature for 3 h. Separately, 59.8 mg of bovine serum albumin or 40.1 mg of ovalbumin was dissolved in 6 mL of phosphate buffer (pH 7.4). The activation solution was added dropwise to the protein solution under ice bath conditions, and the reaction was carried out in the dark for 24 h.

[0073] Protective group removal: Add 4.3 mL of trifluoroacetic acid to adjust the pH to 7.3. The rest is the same as in Example 1.

[0074] Purification was performed in the same manner as in Example 1.

[0075] Complete antigen characterization: Ultraviolet spectroscopy and time-of-flight mass spectrometry identified the coupling ratio of the 3-amino-5-ethoxybenzoic acid hapten to bovine serum albumin as 16.5:1 and to ovalbumin as 13.8:1. Ultraviolet spectroscopy showed a significant change in the maximum absorption peak of the complete antigen compared to the hapten and carrier protein, confirming successful preparation of the complete antigen.

[0076] (3) Antibody preparation This example describes the preparation of polyclonal antibodies. A 1 mg / mL immunogen solution was mixed with an equal volume of carboxylated carbon nanotube dispersion (0.5 mg / mL), and then thoroughly emulsified with Freund's adjuvant at a 1:1 volume ratio until a stable water-in-oil emulsion was formed. New Zealand white rabbits were used as immunization animals, administered via subcutaneous injection at multiple sites, with each rabbit receiving 1 mL per injection. The initial immunization used Freund's complete adjuvant, followed by booster immunizations every two weeks using the same dose of immunogen emulsified with Freund's incomplete adjuvant, for a total of 4-5 immunizations. The final immunization consisted only of immunogen diluted with PBS buffer. Blood was collected from the marginal ear vein 7-10 days after the third immunization, and serum titer was determined using an indirect ELISA method, while inhibition rate was determined using a competitive ELISA method. Rabbits with the best titer and inhibition rate were selected, and blood was collected from their hearts 10-14 days after the final immunization. Serum was separated to obtain the polyclonal antibodies.

[0077] Potency assays showed that the rabbit-derived polyclonal antibody exhibited good specificity and affinity, further confirming the successful synthesis of the artificial antigen and its good immunogenicity. Specificity studies revealed extremely low cross-reactivity with major structural analogs of 3-aminophenethyl ether, indicating no significant cross-reactivity and good specificity. IC50 50With a concentration of 12.45 ng / mL, the antibody exhibits high sensitivity, meeting the requirements for trace detection. Precision evaluation results show that the intra-assay coefficient of variation is less than or equal to 7%, and the inter-assay coefficient of variation is less than or equal to 10%, indicating good repeatability and meeting precision standards for the antibody detection system. In the practical sample suitability evaluation, the sample spiked recoveries ranged from 87.42% to 109.58%, with relative standard deviations ranging from 0.76% to 2.48%. Affinity assay results show that the antibody has a strong binding affinity to 3-aminophenethyl ether, enabling efficient recognition of the target molecule.

[0078] (4) Screening and evaluation of aptamers A library of ssDNA containing 60 random bases was used as the initial library for SELEX screening. Twelve rounds of forward screening were then performed: the first round of incubation was 60 min, with the incubation time decreasing progressively from the second round to 8 min by the twelfth round; simultaneously, the number of high-salt washes was increased from 2 to 8, and the salt concentration was gradually increased from 0.1 mol / L to 0.8 mol / L to progressively improve screening specificity. From the fourth round onwards, reverse screening was introduced: the library was incubated with magnetic beads immobilized with structural analogs at room temperature for 60 min each, and unbound libraries were collected for subsequent forward screening to remove sequences that non-specifically bind to the structural analogs; from the sixth round onwards, an additional round of reverse screening was added, using another structural analog to further eliminate cross-binding sequences. Finally, high-throughput sequencing was performed on the final round of libraries. Candidate sequences were screened by combining sequence and secondary structure prediction to obtain nucleic acid aptamers that can specifically recognize 3-aminophenethyl ether. The sequence was sequenced, and its nucleotide sequence is shown in SEQ ID NO:21, CTATTGAGTCAGGGGGTTGGGGCAACAATAAGCCCTATGCGTGCTACCGTGAAGATGTGC (SEQ ID NO:21).

[0079] The dissociation constant of the aptamer with 3-aminophenethyl ether is 1.6 × 10⁻⁶. -7 It exhibits good affinity and reliable binding ability at mol / L; the cross-reactivity rate with structural analogs is less than 3.2%, demonstrating good specificity. Under conditions of 20℃-40℃ and pH 4.5-8.5, the activity retention rate remains above 90%; it demonstrates strong resistance to matrix interference in actual samples, with a spiked recovery rate above 93%, making it suitable for routine rapid detection.

[0080] (5) Antibody-aptamer conjugation Take 4 μL of antibody and dissolve it in 1 mL of phosphate buffer. Add N-hydroxysuccinimide-biotin (dissolved in dimethyl sulfoxide, concentration 4%) at a molar ratio of antibody to biotin of 1:15. After incubation for 2 h, dialyze to obtain biotinylated antibody.

[0081] The aptamer was modified at the 5' end using the carbodiimide method to obtain the amino-modified aptamer. Separately, 10 nmol of the amino-modified aptamer was dissolved in 500 μL of 0.1 mol / L phosphate buffer (pH 8.0) and mixed with N-hydroxysuccinimide-biotin at a molar ratio of 1:20. After incubation at room temperature for 6 h, the mixture was purified by ultrafiltration to obtain the biotinylated aptamer. The biotinylated antibody was mixed with streptavidin at a molar ratio of 6:1 and incubated at room temperature for 60 min to form a streptavidin-antibody complex. Then, the biotinylated aptamer was added at a ratio of 5:1 (aptamer to initial streptavidin) and incubated at room temperature for 60 min. The reaction solution was purified by gel filtration chromatography to obtain the target antibody-aptamer conjugate.

[0082] (6) Construction of dual-recognition electrochemical sensing interface The gold electrode was pretreated by polishing with 0.05 μm alumina powder, then ultrasonically cleaned in ultrapure water and anhydrous ethanol for 5 min in sequence, and activated by cyclic voltammetry in 1 mol / L sulfuric acid solution (scan range -1.6-0.2 V, scan rate 100 mV / s). After rinsing, it was dried with nitrogen gas to obtain the activated electrode.

[0083] The sensitizing material was modified onto the surface of the activation electrode. 10 μL of GO-rGO / perovskite composite material dispersion with a concentration of 0.8 mg / mL was drop-coated onto the surface of the activation electrode, dried at room temperature for 2 h, rinsed 3 times with phosphate buffer (pH 7.4), and dried with nitrogen to construct the sensing interface.

[0084] Take 10 μL of 0.8% bovine serum albumin solution prepared with phosphate buffer and drop it onto the bottom surface. Incubate at room temperature for 2 h, rinse 4 times with phosphate buffer (pH 7.4), blow dry with nitrogen, and block non-specific binding sites.

[0085] Dilute the antibody-aptamer conjugate to 0.2 mg / mL with phosphate buffer, drop 8 μL onto the blocked substrate surface, incubate at room temperature for 4 h, rinse and dry, add 10 μL of 1% bovine serum albumin solution, block again at room temperature for 30 min, rinse and dry.

[0086] The electrode was placed in a potassium ferrocyanide / potassium ferrocyanide solution and scanned using cyclic voltammetry (scan range -0.2-0.6V, scan rate 40 mV / s) to obtain the final dual-recognition working electrode, labeled BSA / Ab / Apt / GO-rGO / perovskite / AμE.

[0087] (7) Electrochemical characterization Same as Example 1.

[0088] (8) Performance analysis of electrochemical biosensors 12 μL of standard solutions of different concentrations were drop-coated onto the surface of the modified electrode and incubated at 37℃ for 30 min to allow the antigen to fully bind to the electrode surface. After incubation, the electrode was rinsed with 0.1 mol / L phosphate buffer (pH 7.4). Subsequently, the electrode was placed in a 0.1 mol / L potassium chloride test solution containing 5 mmol / L potassium ferrocyanide / potassium ferrocyanide, and differential pulse voltammetry was used for detection. The linear regression equation for egg was Y = 1.1147 + 0.0078X, with a correlation coefficient r of 0.9999 and a detection limit of 0.06 μg / kg. This indicates that the constructed sensor has good sensitivity and a low detection limit, and can be used for the detection of trace amounts of 3-aminophenethyl ether.

[0089] (9) Determination of the recovery rate of spiked egg samples Pretreatment of egg samples: Take 5 g of egg sample and homogenize the whole egg liquid. Add 10 mL of acetonitrile containing 1% formic acid, vortex, then add 4 g of magnesium sulfate and 1 g of sodium chloride, vortex, and then centrifuge at 10000 r / min for 10 min. Collect the supernatant and store it at 4℃. Before detection, dilute the supernatant with 0.01 mol / L phosphate buffer, and then use the prepared electrochemical biosensor to detect the diluted sample solution. The recoveries and relative standard deviations of different spike concentrations in eggs are shown in Table 3. The sample spike recoveries ranged from 86.40% to 99.94%, and the relative standard deviations ranged from 0.75% to 2.78%.

[0090] Table 3. Determination of spiked recovery rate of egg samples

[0091] Example 3

[0092] The difference between this embodiment and Embodiment 1 is that: (1) Synthesis of haptens 3 g of methyl 3-amino-5-ethoxybenzoate was placed in 36 mL of methanol solution, and sodium hydroxide solution was added. The mixture was stirred at room temperature for 18-48 h. After the reaction was complete as detected by TLC, the methanol was removed by concentration, the mixture was diluted with ultrapure water, and concentrated hydrochloric acid was added dropwise to precipitate a solid. The solid was then filtered. The filter cake was washed three times each with water and petroleum ether (30 mL each time), and the solid was collected and dried. The filtrate was extracted with ethyl acetate, and the extracts were combined and concentrated to obtain a solid, which was then dried to obtain the 3-amino-5-ethoxybenzoic acid hapten. The rest of the process was the same as in Example 1.

[0093] (2) Synthesis of complete antigen Amino protection: 8 g of 3-amino-5-ethoxybenzoic acid was dissolved in 10 mL of tetrahydrofuran. 6.32 mL of N,N-dimethylformamide and 5.84 mL of di-tert-butyl dicarbonate were added under ice bath conditions, and the reaction was carried out at room temperature in the dark for 8 h. The reaction solution was poured into 60 mL of ice water, and the pH was adjusted to 4.0 with hydrochloric acid, resulting in the precipitation of a solid. The solid was allowed to stand at 4 °C for 1 h, filtered, and washed with ice water until neutral. The crude product was purified by silica gel column chromatography using dichloromethane-methanol (15:1, v / v) as the eluent to give 3-(tert-butoxycarbonylamino)-5-ethoxybenzoic acid.

[0094] Adding the spacer arm: Dissolve 600 mg of 3-(tert-butoxycarbonylamino)-5-ethoxybenzoic acid in 6 mL of anhydrous dichloromethane. While stirring in an ice bath, add 378.7 mg of 6-aminohexanoic acid and 482.1 mg of 1-hydroxybenzotriazole sequentially, and finally add 683.7 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. Remove the ice bath and react at room temperature in the dark for 18 h. The rest of the reaction is the same as in Example 1.

[0095] Coupling with carrier proteins: Using the carbodiimide method, 20 mg of the above hapten was dissolved in 1.5 mL of anhydrous N,N-dimethylformamide, and 42.6 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 34.1 mg of N-hydroxysuccinimide were added. Activation was carried out at room temperature for 12 h. Separately, 81.4 mg of bovine serum albumin or 54.2 mg of ovalbumin was dissolved in 6 mL of phosphate buffer (pH 7.4). The activation solution was added dropwise to the protein solution under ice bath conditions, and the reaction was carried out in the dark for 16 h.

[0096] Protecting group removal: Add 6.4 mL of trifluoroacetic acid and adjust the pH to 7.4 with 1 mol / L sodium hydroxide solution. The rest is the same as in Example 1.

[0097] Purification was performed in the same manner as in Example 1.

[0098] Characterization of the complete antigen: UV and flight-of-flight mass spectrometry confirmed that the coupling ratio of the 3-amino-5-ethoxybenzoic acid hapten to bovine serum albumin was 15.5:1, and to ovalbumin was 12.6:1. UV spectroscopy showed a significant change in the maximum absorption peak of the complete antigen compared to the hapten and carrier protein, confirming the successful preparation of the complete antigen.

[0099] (3) Antibody preparation This embodiment employs a method of immunizing goats with cationic liposomes encapsulating immunogen and Freund's adjuvant. Cationic lipids and cholesterol (molar ratio 1:1) were accurately weighed and dissolved in chloroform. The organic solvent was removed by vacuum evaporation using a rotary evaporator, resulting in a uniform lipid film on the bottle wall. Phosphate buffer containing immunogen (immunogen concentration 5 mg / mL) was then added for hydration. The lipid film was thoroughly washed away by shaking, and cationic liposomes with uniform particle size were obtained using a probe sonication. After dialysis to remove unencapsulated free antigen, the volume was concentrated to 2 mL, ensuring a final immunogen concentration of not less than 2 mg / mL. One mL of the immunogen-liposome complex was taken and emulsified thoroughly with an equal volume of Freund's complete adjuvant (for primary immunization) or incomplete adjuvant (for booster immunization) by vortexing to form a stable water-in-oil emulsion. Subcutaneous injections were administered at 4-6 points on the rump. Booster immunizations were given every 2 weeks with the same dose of emulsified antigen, for a total of 3-4 immunizations. 10-14 days after the last immunization, blood was collected from the jugular vein to separate serum. The serum antibody titer was measured by indirect ELISA, and the specific inhibition rate against the target hapten was measured by competitive ELISA.

[0100] The results showed that immunization induced goats to produce high-titer, high-specificity polyclonal antibodies, proving that this adjuvant regimen is suitable for immunization with artificial antigens. Specificity experiments showed that the antibodies did not exhibit significant cross-binding to 3-aminophenethyl ether and its various structural analogs, demonstrating outstanding recognition specificity and excellent specificity levels. 50 The concentration was 10.29 ng / mL, demonstrating excellent overall sensitivity. Precision experiments showed that the intra-assay coefficient of variation was no higher than 6%, and the inter-assay coefficient of variation was no higher than 9%, indicating stable and reliable detection results with good reproducibility. This antibody maintained stable recognition and binding ability in actual sample matrices, exhibiting strong resistance to matrix interference and meeting the requirements for practical sample detection.

[0101] (4) Screening and evaluation of aptamers A library of ssDNA containing 80 random bases was used as the initial library for SELEX screening. Sixteen rounds of forward screening were then performed: the first round of incubation was 60 min, and the incubation time was gradually shortened from the second round onwards, reaching 10 min by the tenth round; simultaneously, the number of high-salt washes was increased from 2 to 6, and the salt concentration was gradually increased from 0.1 mol / L to 0.6 mol / L to progressively improve screening specificity. From the sixth round onwards, reverse screening was introduced: before each round of forward screening, the library was incubated sequentially with magnetic beads immobilized with structural analogs at room temperature for 45 min each; unbound libraries were collected for subsequent forward screening to remove sequences that non-specifically bind to structural analogs, thereby improving the specific recognition ability for 3-aminophenethyl ether. Finally, high-throughput sequencing was performed on the final round of libraries. Candidate sequences were screened by combining sequence and secondary structure prediction to obtain nucleic acid aptamers that can specifically recognize 3-aminophenethyl ether. The sequence was sequenced, and its nucleotide sequence is shown in SEQ ID NO:22, AATAAGCCCTATGCGTGCTACCGTGAAGATGTGCTCGAACTATTGAGTCAGGGGGTTGGG (SEQ ID NO:22).

[0102] The dissociation constant of the aptamer with 3-aminophenethyl ether is 2.2 × 10⁻⁶. -7 It exhibits moderate affinity and stable binding at mol / L; cross-reactivity with structural analogs is less than 2.1%, demonstrating excellent specificity. Within the temperature range of 25℃-42℃ and pH 5.0-9.0, the binding activity retention rate exceeds 91%; it maintains accurate recognition capability even in complex matrices, with a spiked recovery rate exceeding 94%, meeting the requirements for practical sample detection applications.

[0103] (5) Antibody-aptamer conjugation Dissolve 1 μL of antibody in 300 μL of phosphate buffer, add N-hydroxysuccinimide-biotin (dissolved in dimethyl sulfoxide, concentration 3%) at a molar ratio of antibody to biotin of 1:12, incubate at room temperature for 1 h and dialyze to obtain biotinylated antibody.

[0104] The aptamer was modified at the 5' end using the carbodiimide method to obtain the amino-modified aptamer. Separately, 5 nmol of the amino-modified aptamer was dissolved in 100 μL of 0.1 mol / L phosphate buffer (pH 8.0) and mixed with N-hydroxysuccinimide-biotin at a molar ratio of 1:16. After incubation at room temperature for 2 h, the mixture was purified by ultrafiltration to obtain the biotinylated aptamer. The biotinylated antibody was mixed with streptavidin at a molar ratio of 2:1 and incubated at room temperature for 30 min to form a streptavidin-antibody complex. Then, the biotinylated aptamer was added at a molar ratio of aptamer to initial streptavidin of 3:1 and incubated at room temperature for 30 min. The reaction solution was purified by filtration chromatography to obtain the target antibody-aptamer conjugate.

[0105] (6) Construction of dual-recognition electrochemical sensing interface The gold electrode was pretreated by polishing with 0.05 μm alumina powder, then ultrasonically cleaned in ultrapure water and anhydrous ethanol for 6 min in sequence, and activated by cyclic voltammetry in 1 mol / L sulfuric acid solution (scan range -1.6-0.2 V, scan rate 100 mV / s). After rinsing, it was dried with nitrogen gas to obtain the activated electrode.

[0106] The sensitizing material was modified onto the surface of the activation electrode. 8 μL of a 1 mg / mL MXene / GO-rGO composite dispersion was drop-coated onto the surface of the activation electrode, dried at room temperature for 2 h, rinsed 3 times with phosphate buffer (pH 7.4), and dried with nitrogen to construct the sensing interface.

[0107] Take 10 μL of 1% bovine serum albumin solution prepared with phosphate buffer and drop it onto the bottom surface. Incubate at room temperature for 3 h, rinse 4 times with phosphate buffer (pH 7.4), blow dry with nitrogen, and block non-specific binding sites.

[0108] Dilute the antibody-aptamer conjugate to 0.3 mg / mL with phosphate buffer, drop 10 μL onto the blocked substrate surface, incubate at room temperature for 4 h, rinse and dry; add 8 μL of 1% bovine serum albumin solution, block again at room temperature for 50 min, rinse and dry.

[0109] The electrode was placed in a potassium ferrocyanide / potassium ferrocyanide solution and scanned using cyclic voltammetry (scan range -0.2-0.6V, scan rate 40 mV / s) to obtain the final dual-recognition working electrode, labeled BSA / Ab / Apt / GO-rGO / perovskite / AμE.

[0110] (7) Electrochemical characterization Same as Example 1.

[0111] (8) Performance analysis of electrochemical biosensors Eight μL of standard solutions of different concentrations were dropped onto the surface of the modified electrode and incubated at 37℃ for 50 min to allow the antigen to fully bind to the electrode surface. After incubation, the electrode was rinsed with 0.1 mol / L phosphate buffer (pH 7.4). Subsequently, the electrode was placed in a 0.1 mol / L potassium chloride test solution containing 5 mmol / L potassium ferrocyanide / potassium ferrocyanide, and differential pulse voltammetry was used for detection. Within the range of 0.5–400 μg / kg, the concentration of 3-aminophenethyl ether showed a linear relationship with ΔI. The linear regression equation was Y = 1.0128 + 0.0069X, with a correlation coefficient r of 0.9999 and a detection limit of 0.08 μg / kg. This indicates that the constructed sensor has good sensitivity and a low detection limit, and can be used for the detection of trace amounts of 3-aminophenethyl ether.

[0112] (9) Determination of spiked recovery rate of chicken liver samples Pretreatment of chicken liver samples: After removing fascia and connective tissue from fresh chicken liver samples, wash with ultrapure water and blot dry. Weigh 5 g of chicken liver tissue and place it in a mortar. Add 10 mL of 0.01 mol / L phosphate buffer solution (pH 7.4) and grind under ice bath conditions to obtain a chicken liver homogenate. Add 3 mL of 10% trichloroacetic acid solution and vortex thoroughly to mix evenly. Then centrifuge at 10000 r / min for 10 min, collect the supernatant and store it at 4℃. Before detection, dilute the supernatant with 0.01 mol / L phosphate buffer solution, and then use the prepared electrochemical biosensor to detect the diluted sample solution. The recoveries and relative standard deviations of different spike concentrations in chicken liver are shown in Table 4. The sample spike recoveries ranged from 83.78% to 101.00%, and the relative standard deviations ranged from 0.97% to 3.16%.

[0113] Table 4. Determination of spiked recovery rate in chicken liver samples.

[0114] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a dual-recognition electrochemical biosensor, characterized in that: Includes the following steps: Step 1: Using 3-amino-5-ethoxybenzoic acid as a hapten, the 3-amino-5-ethoxybenzoic acid hapten was obtained after ester hydrolysis and purification. Step 2: The hapten obtained in Step 1 is sequentially protected with amino groups, added to a spacer arm, coupled with a carrier protein, deprotected, and purified to obtain the complete antigen. Step 3: Using the complete antigen obtained in Step 2 as an immunogen, immunize the animals to prepare specific antibodies; the antibodies are monoclonal antibodies or polyclonal antibodies. Step 4: Using 3-aminophenethyl ether as a target, nucleic acid aptamers that specifically recognize 3-aminophenethyl ether are screened using SELEX technology. The screening includes forward screening and reverse screening steps. Step 5: The antibody obtained in step 3 is conjugated with the aptamer obtained in step 4 using the biotin-streptavidin method to prepare antibody-aptamer conjugates. Step 6: Modify the surface of the gold electrode with nano-sensitizing material to form a signal amplification substrate; add bovine serum albumin solution for the first blocking; dilute the antibody-aptamer conjugate obtained in step 5 with phosphate buffer and add it to the blocked substrate surface for incubation. A second blocking process was performed by adding bovine serum albumin solution again. After interface activation, the dual-recognition electrochemical sensing interface is constructed.

2. The construction method according to claim 1, characterized in that: In step 3, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:1, SEQ ID NO:5 or SEQ ID NO:9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:3, SEQ ID NO:7 or SEQ ID NO:11; the nucleotide coding sequences of the heavy chain variable region are shown in SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10, and the nucleotide coding sequences of the light chain variable region are shown in SEQ ID NO:4, SEQ ID NO:8 or SEQ ID NO:12, respectively. The PCR amplification primers for the monoclonal antibody variable region gene are selected from any one or more of the following primer pairs: the nucleotide sequences of the upstream primers VH-F1 to VH-F10 of the heavy chain variable region are shown in SEQ ID NO:23 to SEQ ID NO:32, respectively; the nucleotide sequences of the downstream primers VH-R1 to VH-R10 of the heavy chain variable region are shown in SEQ ID NO:33 to SEQ ID NO:42, respectively; the nucleotide sequences of the upstream primers VL-F1 to VL-F10 of the light chain variable region are shown in SEQ ID NO:43 to SEQ ID NO:52, respectively; and the nucleotide sequences of the downstream primers VL-R1 to VL-R10 of the light chain variable region are shown in SEQ ID NO:53 to SEQ ID NO:62, respectively.

3. The construction method according to claim 1, characterized in that: In step 4, the SELEX screening includes: pre-cleaning with blank epoxy magnetic beads, followed by 10-18 rounds of forward screening, with the incubation time shortened, the number of washes increased, and the salt concentration increased in each round. From the 3rd to the 9th round, structural analogs are introduced for reverse screening. After each round of screening, ssDNA libraries are prepared by PCR amplification and λ exonuclease digestion. Finally, nucleic acid aptamers are obtained by sequencing and structural prediction. The nucleotide sequence of the nucleic acid aptamer is any one of SEQ ID NO:13 to SEQ ID NO:

22.

4. The construction method according to claim 1, characterized in that: The synthesis of the hapten in step 1 specifically includes the following steps: using 1-3 g of methyl 3-amino-5-ethoxybenzoate as raw material, dissolving it in 12-36 mL of organic solvent, adding 1 mol / L-4 mol / L sodium hydroxide solution to adjust the pH to 5.0-8.0, extracting with ethyl acetate, and washing the extract phase three times each with 10-30 mL of water and petroleum ether to obtain the purified 3-amino-5-ethoxybenzoic acid hapten.

5. The construction method according to claim 1, characterized in that: The synthesis of the complete antigen in step 2 specifically includes the following steps: Step 2a, amino protection: Dissolve 3-8 g of 3-amino-5-ethoxybenzoic acid in 6-16 mL of tetrahydrofuran, add 2.37-6.32 mL of N,N-dimethylformamide and 2.19-5.84 mL of di-tert-butyl dicarbonate in an ice bath, and react at room temperature in the dark for 5-10 h; pour the reaction solution into 40-80 mL of ice water, adjust the pH to 3.0-4.0 with hydrochloric acid, and a solid precipitates; let stand at 4℃ for 1-2 h, filter, wash with ice water until neutral; purify by silica gel column chromatography with dichloromethane / methanol as eluent, collect the product and evaporate to dryness by rotary evaporation to obtain 3-(tert-butoxycarbonylamino)-5-ethoxybenzoic acid; Step 2b, adding the spacer arm: Take 200-600 mg of the product obtained in step 2a, dissolve it in 4-12 mL of anhydrous dichloromethane, and add 126.7-380.1 mg of the spacer arm, 160.7-482.1 mg of 1-hydroxybenzotriazole and 227.9-683.7 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride sequentially with stirring in an ice bath. Remove the ice bath and react at room temperature in the dark for 10-18 h. After the reaction is complete, concentrate to remove dichloromethane, dissolve the residue in ethyl acetate or dichloromethane, wash successively with 0.1 mol / L dilute hydrochloric acid and water, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. Step 2c, coupling with the carrier protein: Using the carbodiimide method, take 12-25 mg of the product obtained in step 2b, dissolve it in 0.6-1.8 mL of N,N-dimethylformamide, add 24.75-51.9 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 22.4-46.7 mg of N-hydroxysuccinimide, and activate at room temperature for 2-4 h; separately, dissolve 28.8-81.4 mg of the carrier protein in 4-12 mL of phosphate buffer at pH 7.4, add the activation solution dropwise to the protein solution under ice bath, and react at 4°C in the dark for 12-24 h; the carrier protein is bovine serum albumin or ovalbumin; Step 2d, Deprotection of protecting group: After the coupling reaction is completed, 2.5-6.4 mL of trifluoroacetic acid is added dropwise to the reaction solution under ice bath conditions. The reaction is stirred under ice bath conditions for 1 h in the dark. Then, the pH is adjusted to 7.2-7.4 with 1 mol / L sodium hydroxide solution under ice bath conditions to terminate the reaction. Step 2e, purification: Transfer the reaction solution to a dialysis bag and dialyze it in 0.01 mol / L pH 7.4 phosphate buffer at 4°C for 24-72 h. After dialysis, transfer the solution in the bag to a centrifuge tube and centrifuge at 10000 r / min at 4°C for 10 min. Collect the supernatant to obtain the complete antigen.

6. The construction method according to claim 1, characterized in that: The preparation method of the antibody-aptamer conjugate in step 5 is as follows: An amino-modified antibody is reacted with N-hydroxysuccinimide-biotin reagent at a molar ratio of 1:10-20 to obtain a biotinylated antibody; an amino-modified aptamer is reacted with N-hydroxysuccinimide-biotin reagent at a molar ratio of 1:10-25 to obtain a biotinylated aptamer; the biotinylated antibody is mixed with streptavidin at a molar ratio of 2-6:1 and incubated at room temperature to form a streptavidin-antibody complex; then, the biotinylated aptamer is added at a molar ratio of aptamer to streptavidin of 2-5:1, incubated at room temperature, and purified by gel filtration chromatography to obtain the final product.

7. The construction method according to claim 1, characterized in that: The nano-sensitizing material mentioned in step 6 is any one of MXene / AuNPs, MXene / GO-rGO, or GO-rGO / perovskite; the perovskite is any one of CsPbX3, MAPbX3, FAPbX3, or Cs2AgBiBr6.

8. An electrochemical aptamer-antibody dual recognition sensor prepared by the construction method according to any one of claims 1-7.

9. The application of the sensor according to claim 8 in detecting 3-aminophenethyl ether in animal-derived foods, characterized in that: Includes the following steps: Step 1, Sample pretreatment: Take animal food samples, mince or homogenize them, add extraction reagent for extraction, precipitate protein with sodium sulfate, centrifuge, collect the supernatant, dilute with phosphate buffer to obtain sample treatment solution; Step 2: Contact the working electrode of the electrochemical aptamer-antibody dual recognition sensor described in claim 8 with the sample processing solution obtained in step 1, and incubate it under constant temperature conditions to allow 3-aminophenethyl ether in the sample to bind to the recognition element on the electrode surface. Step 3: After incubation, clean the electrode and place it in potassium chloride test solution containing potassium ferrocyanide / potassium ferrocyanide. Use differential pulse voltammetry to detect the oxidation peak current value I. Step 4: Using the oxidation peak current value of the blank solution as I0, calculate the current change ΔI = I0 - I. Substitute ΔI into the standard curve showing a linear relationship between the concentration of 3-aminophenethyl ether and ΔI to calculate the content of 3-aminophenethyl ether in the animal food sample.

10. The application according to claim 9, characterized in that: The animal-derived food mentioned in step 1 is any one of eggs, chicken meat, chicken liver, or chicken kidney; the extraction reagent is any one of methanol, acetonitrile, n-hexane, or a trichloroacetic acid-acetonitrile mixture, wherein the volume ratio of trichloroacetic acid to acetonitrile in the trichloroacetic acid-acetonitrile mixture is 1:5-10.

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