Noxalagin detection card and preparation method thereof

Abstract

The invention belongs to the technical field of immunodetection, and particularly relates to a norakaurin detection card and a preparation method thereof.The norakaurin detection card comprises a card shell, a PVC bottom plate is fixedly installed at the bottom end of an inner cavity of the card shell, and the top of the PVC bottom plate is sequentially in lap joint with a fixed sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad from left to right; the combination pad is coated with a colloidal gold labeled noraudin monoclonal antibody-nano silver composite probe, and the nitrocellulose membrane is provided with a detection line and a quality control line. The nano-silver modified colloidal gold particles are adopted to prepare the composite probe, the optical signal transduction efficiency of the probe is optimized, the signal response of a low-concentration target object is enhanced, and the composite probe has the advantages that accurate capture of low-concentration norakaurin residues is achieved, the detection sensitivity is greatly improved, and the trace residue detection requirement is met.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, specifically to a nocoagulant detection card and its preparation method. Background Technology

[0002] Nocturia, a broad-spectrum antibacterial feed additive, is widely used in the livestock and poultry farming industry. It effectively inhibits the growth of pathogenic bacteria and improves survival rates and feed conversion ratios. However, nocturia leaves residues in livestock and poultry. Long-term consumption of livestock and poultry products containing nocturia residues can pose potential health risks to humans, such as disrupting intestinal flora balance and inducing the development of drug-resistant strains. Therefore, establishing a rapid, accurate, and convenient method for detecting nocturia residues is crucial for ensuring food safety and promoting the standardized development of the industry.

[0003] Currently, the main detection methods for nifedipine include high performance liquid chromatography, gas chromatography-mass spectrometry, and traditional immunochromatographic detection. While high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) offer high detection accuracy, they suffer from complex sample pretreatment, long detection cycles (typically 2-4 hours), and reliance on large precision instruments and skilled operators, making them unsuitable for rapid on-site testing. Traditional immunochromatographic assay cards (such as colloidal gold assay cards), while offering advantages like ease of operation and rapid detection, suffer from the following core defects: First, low detection sensitivity, with a detection rate of less than 60% for low concentrations of noracin residues (e.g., below 5 ng / mL), leading to a high likelihood of false negatives. Second, poor specificity, easily exhibiting cross-reactions with structurally similar antibacterial drugs such as tetracyclines and penicillins, with cross-reaction rates exceeding 30%. Third, poor stability, with colloidal gold particles prone to aggregation; after storage at room temperature for more than 3 months, the detection performance decreases by more than 40%. Fourth, narrow sample applicability, easily leading to matrix interference in high-protein, high-fat livestock and poultry tissue samples, resulting in significant deviations in test results. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A nocoagulant detection card includes a card shell, a PVC base plate fixedly installed at the bottom of the inner cavity of the card shell, and a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad sequentially overlapped and fixed on the top of the PVC base plate from left to right. The conjugate pad is coated with a colloidal gold-labeled nocoagulant monoclonal antibody-nano silver composite probe, and the nitrocellulose membrane is provided with a detection line and a quality control line.

[0005] As a preferred embodiment of the novacrine detection card of the present invention, the detection line is coated with novacrine hapten-bovine serum albumin conjugate, and the control line is coated with goat anti-mouse IgG antibody.

[0006] As a preferred embodiment of the Nocogenin test card of the present invention, the sample pad is soaked in a composite pretreatment solution, which includes Tris-HCl buffer, Tween-20, bovine serum albumin and disodium EDTA.

[0007] As a preferred embodiment of the novel alpha-glucanase detection card of the present invention, the card shell has a sample application hole on the top left side, and the sample application hole is located directly above the sample pad.

[0008] As a preferred embodiment of the Nocogenin test card of the present invention, an observation window is provided on the top right side of the card shell, and the observation window is located directly above the nitrocellulose membrane.

[0009] A method for preparing a nocoagulant detection card includes the following specific steps: Preparation of S1, Novogene monoclonal antibody: S11, Hapten Synthesis: Using nifedipine as a raw material, succinic anhydride was added as a modifier and the reaction was carried out at a constant temperature in a pyridine solvent. After the reaction was completed, deionized water was added for dilution, and the pH was adjusted to 2.0-3.0 with hydrochloric acid to precipitate the precipitate. The precipitate was washed three times with diethyl ether and then purified by silica gel column chromatography. The target component was collected and vacuum dried to obtain the nifedipine hapten. S12, Immunogen Preparation: Take the nifedipine hapten prepared in S11, add N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and activate at room temperature for 20-30 minutes to obtain the hapten activation solution; dissolve bovine serum albumin (BSA) in PBS buffer (pH 7.4), and slowly add it dropwise to the hapten activation solution while stirring; after the reaction is complete, dialyze with PBS buffer for 3 days, changing the dialysate 3 times a day to remove unbound hapten, and obtain the nifedipine hapten-BSA conjugate (immunogen). S13, Animal Immunization: 6-8 week old female BALB / c mice were selected. The novacrine hapten-BSA conjugate prepared in S12 was mixed with an equal volume of Freund's complete adjuvant and emulsified, and then injected intraperitoneally. The immunization dose was 50 μg / mouse. After the first immunization, booster immunizations were performed every 2 weeks. For booster immunizations, the novacrine hapten-BSA conjugate was mixed with an equal volume of Freund's incomplete adjuvant and emulsified, and the immunization dose was 30 μg / mouse. A total of 3 booster immunizations were performed. Seven days after the last immunization, tail vein blood was collected from the mice, and serum antibody titers were detected by indirect ELISA. Mice with antibody titers higher than 1:100000 were selected as donors. S14, Hybridoma Cell Preparation and Screening: Spleens from mice with qualified antibody titers in S13 were collected, ground, and splenocytes were obtained. Splenocytes were mixed with myeloma cells (SP2 / 0) at a ratio of 5:1, and polyethylene glycol (PEG4000) was added for fusion. After fusion, the cells were seeded into 96-well cell culture plates containing HAT selective medium and incubated at 37°C with 5% [temperature missing]. Cultured in an incubator; after 7-10 days of culture, positive wells were screened by indirect ELISA, and the cells in the positive wells were subjected to three limiting dilution clonal culture to finally obtain hybridoma cell lines that can stably secrete norostatin monoclonal antibodies; S15, Monoclonal Antibody Purification: The hybridoma cell line obtained in S14 was inoculated into the peritoneal cavity of mice to induce ascites; after collecting the ascites, it was purified using a Protein G affinity chromatography column. The chromatography column was first equilibrated with equilibration buffer, then the ascites was loaded, the target antibody was eluted with elution buffer, the elution peak was collected and dialyzed to desalt, and high-purity nifedipine monoclonal antibody was obtained. S2, Preparation of colloidal gold-labeled novolucretin monoclonal antibody-silver nanoparticle composite probe: S21, Preparation of colloidal gold particles: Using the sodium citrate reduction method, 100 mL of 0.01% chloroauric acid solution was placed in a three-necked flask and heated to boiling. 10 mL of 1% sodium citrate solution was quickly added and the mixture was boiled for 15-20 minutes. The solution gradually changed from pale yellow to wine red. Heating was stopped and the solution was cooled to room temperature to obtain a colloidal gold solution with a particle size of 20-30 nm. S22, Silver-modified colloidal gold particles: Take the colloidal gold solution prepared in S21, and use 0.1 mol / L... The pH of the solution was adjusted to 8.5-9.0, 10 mL of 0.05% silver nitrate solution was added, and after stirring evenly, 5 mL of 0.1% sodium borohydride solution was slowly added dropwise. The reaction was carried out at room temperature in the dark with stirring for 1 hour to obtain colloidal gold nanoparticles modified with silver nanoparticles. After the reaction was completed, the supernatant was discarded after centrifugation, and the precipitate was resuspended in PBS buffer (pH 7.4) containing 0.02% sodium azide to obtain a solution of silver nanoparticle-colloidal gold composite particles. S23, Composite Probe Labeling: Take the nano-silver-colloidal gold composite particle solution prepared in S22, add the purified nifedipine monoclonal antibody from S15, the antibody addition amount is 50 μg / mL, and stir at room temperature in the dark for 1 hour; add bovine serum albumin (BSA) to the final concentration of 1%, continue stirring for 30 minutes to block the unbound active sites; then centrifuge at 12000 r / min for 30 minutes, discard the supernatant, resuspend the precipitate in PBS buffer (pH 7.4) containing 0.5% Tween-20, 1% BSA and 0.02% sodium azide, and adjust the probe concentration to OD520nm=1.5 to obtain the colloidal gold-labeled nifedipine monoclonal antibody-nano-silver composite probe; S24, Crosslinking and Curing Modification of Composite Probe: Take the composite probe solution prepared in S23, add glutaraldehyde crosslinking agent to a final concentration of 0.2%, and stir at room temperature in the dark for 40 minutes; utilize the bifunctional characteristics of glutaraldehyde to initially activate the active sites on the probe surface; add 0.5% polyethyleneimine (PEI) to the reaction solution and continue stirring for 20 minutes to form a stable structure of probe-PEI-crosslinking agent, enhancing the adsorption force between the probe and the binding pad substrate; add glycine to a final concentration of 1% and stir for 15 minutes to block unreacted crosslinking sites and avoid subsequent nonspecific binding; centrifuge at 12000 r / min for 30 minutes, discard the supernatant, and resuspend the precipitate with the original probe resuspending buffer to obtain the crosslinked and cured composite probe; S3, Preparation of core components of the detection card: S31, Sample pad pretreatment: Take a glass fiber membrane as the sample pad substrate, immerse it in the composite pretreatment solution for 30 minutes; after immersion, place it in an oven to dry for later use. S32, Hydrophilicity-Hypersensitivity Gradient Modification of Nitrocellulose Membranes: A gradient modification solution was prepared, consisting of Tris-HCl buffer solutions (pH 8.0) containing 0.1%, 0.3%, and 0.5% Tween-80, respectively, forming a hydrophilicity gradient from low to high. The nitrocellulose membrane was laid flat on the modification platform, and the dip-pull method was used. First, the end of the nitrocellulose membrane near the binding pad was immersed in a 0.5% Tween-80 solution (strong hydrophilicity, facilitating probe migration), and the end near the absorbent pad was immersed in a 0.1% Tween-80 solution (weak hydrophilicity, preventing band diffusion). The middle region was transitioned by a gradient in the pulling speed (2 mm / s → 1 mm / s). The modified nitrocellulose membrane was then placed in an oven to dry and solidify the gradient structure. S33, Preparation of bonding pad: A polyester film is used as the substrate for the bonding pad. The cross-linked and cured composite probe prepared in S24 is uniformly sprayed onto the polyester film at a spraying amount of 8 μL / cm². After spraying, the film is placed in a vacuum drying oven for drying. After drying, it is sealed and stored in a dry environment for later use. S34, Nitrocellulose membrane treatment: Take the nitrocellulose membrane modified by S32, and use a membrane scrubbing instrument to divide the nitrocellulose membrane into a test line (T line) and a control line (C line) with the concentration of 1 mg / mL of the nitrocellulose hapten-bovine serum albumin conjugate and the goat anti-mouse IgG antibody (2 mg / mL). The distance between the test line and the control line is 5 mm, and the scrubbing speed is 3 cm / s. After scrubbing, place the membrane in an oven to dry for later use. S35, Pre-treatment of absorbent pad: Take absorbent paper as the base material for the absorbent pad, cut it to the size that matches the PVC base plate, place it in a 120℃ oven to dry for 30 minutes to remove moisture, and set aside. S4: Assembly of the detection card: S41, Component overlap: Take a PVC base plate and, in the order of sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad, overlap and fix the sample pad pretreated in S31, the conjugate pad prepared in S33, the nitrocellulose membrane treated in S34, and the absorbent pad pretreated in S35 onto the PVC base plate in sequence; wherein, the right end of the conjugate pad overlaps the left end of the NC membrane (overlap length is 2mm), the right end of the NC membrane overlaps the left end of the absorbent pad (overlap length is 2mm), the left end of the sample pad is aligned with the left end of the PVC base plate, and the right end of the absorbent pad is aligned with the right end of the PVC base plate; S42, Clamp assembly: Install the completed structure from S41 into the clamp, ensuring that the sample pad corresponds to the sample application port of the clamp, and that the detection line and control line of the nitrocellulose membrane correspond to the observation window of the clamp; after assembly, use a tablet press to compact the clamp, ensuring that all components are firmly fixed.

[0010] In a preferred embodiment of the preparation method of the Nocogenin test card according to the present invention, in step S11, the reaction temperature in the pyridine solvent is set to 40-45°C and the reaction time is set to 3-4 hours; in step S12, the reaction temperature in the hapten activation solution is set to 4°C and the reaction time is set to 12-16 hours.

[0011] In a preferred embodiment of the preparation method of the Nocogenin detection card of the present invention, in step S22, after the reaction is completed, the centrifugation speed is set to 10000 r / min and the centrifugation time is set to 20 minutes.

[0012] As a preferred embodiment of the preparation method of the Nocogenin test card of the present invention, in step S31, the raw materials of the composite pretreatment solution include, by percentage, 20 mmol / L Tris-HCl buffer, 0.5% Tween-20, 2% bovine serum albumin, and 0.1% disodium EDTA, and the drying temperature after soaking is set to 37°C and the drying time is set to 2 hours.

[0013] In a preferred embodiment of the preparation method of the Nocogenin test card according to the present invention, in step S32, the drying temperature of the oven is set to 37°C and the drying time is set to 2 hours; in step S33, the drying temperature of the vacuum drying oven is set to 37°C and the drying time is set to 1 hour; in step S34, the drying temperature of the oven is set to 37°C and the drying time is set to 3 hours.

[0014] Compared with existing technologies: 1. High sensitivity: By using nano-silver to modify colloidal gold particles to prepare composite probes, the optical signal transmission efficiency of the probes is optimized, and the signal response of low-concentration target substances is enhanced. It has the advantages of accurately capturing low-concentration noraquinone residues, greatly improving detection sensitivity, and meeting the needs of trace residue detection. 2. High specificity: By refining the molecular modification steps of hapten synthesis and optimizing the immunogen preparation and hybridoma cell screening process, the targeting recognition ability of monoclonal antibodies is improved. This has the advantages of accurately distinguishing novolucretin from structurally similar antibacterial drugs, avoiding cross-reactions, and reducing false positive results. 3. Good stability: By performing BSA sealing treatment on the composite probe and optimizing the sample pad with composite pretreatment solution, the influence of the external environment on the core components can be reduced, and the detection card can maintain stable detection performance after long-term storage, avoiding significant performance degradation. 4. Wide sample applicability: By adding disodium EDTA and bovine serum albumin to the composite pretreatment solution, metal ion chelation and impurity protein adsorption are achieved respectively, reducing interference from complex matrices. It has the advantage of being adaptable to various sample types such as feed, livestock and poultry blood, muscle, and liver, thus broadening the detection scenarios. 5. Simple operation and fast detection: By optimizing the structural design and component compatibility of the test card, the sample pretreatment process is simplified. It does not require large precision instruments and professional operating skills. It has the advantages of enabling rapid sample addition and reading results in a short time, which is suitable for on-site real-time detection needs. 6. Secure probe fixation: By subjecting the composite probe to dual treatment of glutaraldehyde crosslinking and PEI modification, and then blocking unreacted active sites with glycine, the interaction between the probe and the binding pad substrate is strengthened. This has the advantage of avoiding signal abnormalities caused by probe detachment during detection and further improving detection stability. 7. High strip clarity: By modifying the NC membrane with gradient concentration Tween-80 solution and precisely controlling the lifting speed to construct a hydrophilic-hydrophobic gradient, the liquid migration path and rate are optimized. This has the advantages of avoiding strip distortion, diffusion and tailing, improving T / C line recognition, and facilitating fast and accurate interpretation. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the structure of the present invention; Figure 2 This is a front view schematic diagram of the structure of the present invention; Figure 3 This is a top view of the structure of the present invention.

[0016] In the diagram: 10 cassette, 11 sample loading hole, 12 observation window, 20 PVC base plate, 30 sample pad, 40 conjugate pad, 50 nitrocellulose membrane, 51 detection line, 52 quality control line, 60 absorbent pad. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. Example 1:

[0018] This invention provides a norepinephrine detection card; please refer to [link / reference]. Figures 1-3 The device includes a housing 10, with a PVC base plate 20 fixedly installed at the bottom of the inner cavity of the housing 10. The top of the PVC base plate 20 is sequentially overlapped and fixed with a sample pad 30, a conjugate pad 40, a nitrocellulose membrane 50, and an absorbent pad 60 from left to right. The conjugate pad 40 is coated with a colloidal gold-labeled noraconazole monoclonal antibody-nano silver composite probe. The nitrocellulose membrane 50 is provided with a detection line 51 and a control line 52.

[0019] The detection line 51 is coated with a novogene hapten-bovine serum albumin conjugate, and the control line 52 is coated with goat anti-mouse IgG antibody; the sample pad 30 is soaked in a composite pretreatment solution containing Tris-HCl buffer, Tween-20, bovine serum albumin and disodium EDTA.

[0020] The top left side of the casing 10 has a sample application hole 11, which is located directly above the sample pad 30; the top right side of the casing 10 has an observation window 12, which is located directly above the nitrocellulose membrane 50.

[0021] A method for preparing a nocoagulant detection card includes the following specific steps: Preparation of S1, Novogene monoclonal antibody: S11, Hapten Synthesis: Using nifedipine as a raw material, succinic anhydride was added as a modifier. The reaction was carried out in pyridine solvent at 40°C for 3 hours. After the reaction, deionized water was added for dilution, and the pH was adjusted to 2.0 with hydrochloric acid to precipitate the precipitate. The precipitate was washed three times with ether and purified by silica gel column chromatography. The target component was collected and vacuum dried to obtain the nifedipine hapten. S12, Immunogen Preparation: Take the nifedipine hapten prepared in S11, add N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and activate at room temperature for 20 minutes to obtain the hapten activation solution; dissolve bovine serum albumin (BSA) in PBS buffer (pH 7.4) and slowly add it dropwise to the hapten activation solution, and stir at 4°C for 12 hours; after the reaction, dialyze with PBS buffer for 3 days, changing the dialysate 3 times a day to remove unbound hapten, and obtain the nifedipine hapten-BSA conjugate (immunogen). S13, Animal Immunization: Six-week-old female BALB / c mice were selected. The novacrine hapten-BSA conjugate prepared in S12 was mixed with an equal volume of Freund's complete adjuvant and emulsified, and then injected intraperitoneally for immunization. The immunization dose was 50 μg / mouse. After the first immunization, booster immunizations were performed every 2 weeks. For booster immunizations, the novacrine hapten-BSA conjugate was mixed with an equal volume of Freund's incomplete adjuvant and emulsified, and the immunization dose was 30 μg / mouse. A total of 3 booster immunizations were performed. Seven days after the last immunization, tail vein blood was collected from the mice, and serum antibody titers were detected by indirect ELISA. Mice with antibody titers higher than 1:100000 were selected as donors. S14, Hybridoma Cell Preparation and Screening: Spleens from mice with qualified antibody titers in S13 were collected, ground, and splenocytes were obtained. Splenocytes were mixed with myeloma cells (SP2 / 0) at a ratio of 5:1, and polyethylene glycol (PEG4000) was added for fusion. After fusion, the cells were seeded into 96-well cell culture plates containing HAT selective medium and incubated at 37°C with 5% [temperature missing]. The cells were cultured in an incubator. After 7 days of culture, positive wells were screened by indirect ELISA. The cells in the positive wells were then subjected to three limiting dilution clonal culture to finally obtain hybridoma cell lines that could stably secrete norostatin monoclonal antibodies. S15, Monoclonal Antibody Purification: The hybridoma cell line obtained in S14 was inoculated into the peritoneal cavity of mice to induce ascites; after collecting the ascites, it was purified using a Protein G affinity chromatography column. The chromatography column was first equilibrated with equilibration buffer, then the ascites was loaded, the target antibody was eluted with elution buffer, the elution peak was collected and dialyzed to desalt, and high-purity nifedipine monoclonal antibody was obtained. S2, Preparation of colloidal gold-labeled novolucretin monoclonal antibody-silver nanoparticle composite probe: S21, Preparation of colloidal gold particles: Using the sodium citrate reduction method, 100 mL of 0.01% chloroauric acid solution was placed in a three-necked flask and heated to boiling. 10 mL of 1% sodium citrate solution was quickly added and the mixture was boiled for another 15 minutes. The solution gradually changed from pale yellow to wine red. Heating was stopped and the mixture was cooled to room temperature to obtain a colloidal gold solution with a particle size of 20 nm. S22, Silver-modified colloidal gold particles: Take the colloidal gold solution prepared in S21, and use 0.1 mol / L... The pH of the solution was adjusted to 8.5, 10 mL of 0.05% silver nitrate solution was added, and after stirring, 5 mL of 0.1% sodium borohydride solution was slowly added dropwise. The reaction was carried out at room temperature in the dark for 1 hour to obtain colloidal gold nanoparticles modified with silver nanoparticles. After the reaction was completed, the mixture was centrifuged at 10000 r / min for 20 minutes, the supernatant was discarded, and the precipitate was resuspended in PBS buffer (pH 7.4) containing 0.02% sodium azide to obtain a solution of silver nanoparticle-colloidal gold composite particles. S23, Composite Probe Labeling: Take the nano-silver-colloidal gold composite particle solution prepared in S22, add the purified nifedipine monoclonal antibody from S15, the antibody addition amount is 50 μg / mL, and stir at room temperature in the dark for 1 hour; add bovine serum albumin (BSA) to the final concentration of 1%, continue stirring for 30 minutes to block the unbound active sites; then centrifuge at 12000 r / min for 30 minutes, discard the supernatant, resuspend the precipitate in PBS buffer (pH 7.4) containing 0.5% Tween-20, 1% BSA and 0.02% sodium azide, and adjust the probe concentration to OD520nm=1.5 to obtain the colloidal gold-labeled nifedipine monoclonal antibody-nano-silver composite probe; S24, Crosslinking and Curing Modification of Composite Probe: Take the composite probe solution prepared in S23, add glutaraldehyde crosslinking agent to a final concentration of 0.2%, and stir at room temperature in the dark for 40 minutes; utilize the bifunctional characteristics of glutaraldehyde to initially activate the active sites on the probe surface; add 0.5% polyethyleneimine (PEI) to the reaction solution and continue stirring for 20 minutes to form a stable structure of probe-PEI-crosslinking agent, enhancing the adsorption force between the probe and the binding pad substrate; add glycine to a final concentration of 1% and stir for 15 minutes to block unreacted crosslinking sites and avoid subsequent nonspecific binding; centrifuge at 12000 r / min for 30 minutes, discard the supernatant, and resuspend the precipitate with the original probe resuspending buffer to obtain the crosslinked and cured composite probe; S3, Preparation of core components of the detection card: S31, Sample pad 30 pretreatment: Take a glass fiber membrane as the sample pad substrate and immerse it in a composite pretreatment solution for 30 minutes; the composite pretreatment solution consists of 20 mmol / L Tris-HCl buffer (pH 8.0), 0.5% Tween-20, 2% bovine serum albumin and 0.1% disodium EDTA; after immersion, place it in a 37℃ oven to dry for 2 hours, and set aside for later use; S32, Hydrophilic-hydrophobic gradient modification of nitrocellulose membrane 50: A gradient modification solution was prepared by adding Tris-HCl buffer (pH 8.0) containing 0.1%, 0.3%, and 0.5% Tween-80 to form a hydrophilic gradient from low to high. The nitrocellulose membrane 50 was laid flat on the modification platform. Using the dip-pull method, the end of the nitrocellulose membrane 50 near the binding pad 40 was first immersed in a 0.5% Tween-80 solution (strong hydrophilicity, facilitating probe migration), and the end near the absorbent pad 60 was immersed in a 0.1% Tween-80 solution (weak hydrophilicity, preventing band diffusion). The middle region was transitioned by a gradient in the pulling speed (2 mm / s → 1 mm / s). The modified nitrocellulose membrane 50 was then placed in a 37°C oven for 2 hours to solidify the gradient structure. S33, Preparation of bonding pad 40: A polyester film was used as the bonding pad substrate. The cross-linked and cured composite probe prepared in S24 was uniformly sprayed onto the polyester film at a spraying amount of 8 μL / cm². After spraying, the film was placed in a vacuum drying oven at 37℃ for 1 hour. After drying, it was sealed and stored in a dry environment for later use. S34, Nitrocellulose Membrane 50 Treatment: Take the S32 modified nitrocellulose membrane 50 and use a membrane scrubbing instrument to divide the nitrocellulose membrane 50 into a detection line 51 (T line) and a control line 52 (C line) with the concentration of 1 mg / mL of the nitrocellulose hapten-bovine serum albumin conjugate and goat anti-mouse IgG antibody (2 mg / mL). The distance between the detection line 51 and the control line 52 is 5 mm, and the scrubbing speed is 3 cm / s. After scrubbing, place the membrane in a 37℃ oven to dry for 3 hours for later use. S35, Pre-treatment of absorbent pad 60: Take absorbent paper as the substrate of absorbent pad, cut it to the size that matches the PVC base plate 20, place it in a 120℃ oven to dry for 30 minutes to remove moisture, and set aside. S4: Assembly of the detection card: S41, Component overlap: Take a PVC base plate 20 and, in the order of sample pad 30, binding pad 40, nitrocellulose membrane 50, and absorbent pad 60, overlap and fix the sample pad 30 (pretreated in S31), binding pad 40 (prepared in S33), nitrocellulose membrane 50 (treated in S34), and absorbent pad 60 (pretreated in S35) onto the PVC base plate 20 in sequence; wherein, the right end of the binding pad overlaps the left end of the NC membrane (overlap length is 2mm), the right end of the NC membrane overlaps the left end of the absorbent pad (overlap length is 2mm), the left end of the sample pad is aligned with the left end of the PVC base plate, and the right end of the absorbent pad is aligned with the right end of the PVC base plate; S42, cassette assembly: Install the overall structure completed in S41 into the cassette 10, ensuring that the sample pad 30 corresponds to the sample application hole 11 of the cassette 10, and that the detection line 51 and quality control line 52 of the nitrocellulose membrane 50 correspond to the observation window 12 of the cassette 10; after assembly, use a tablet press to compact the cassette, ensuring that all components are firmly fixed. Example 2:

[0022] This invention provides a norepinephrine detection card; please refer to [link / reference]. Figures 1-3 The device includes a housing 10, with a PVC base plate 20 fixedly installed at the bottom of the inner cavity of the housing 10. The top of the PVC base plate 20 is sequentially overlapped and fixed with a sample pad 30, a conjugate pad 40, a nitrocellulose membrane 50, and an absorbent pad 60 from left to right. The conjugate pad 40 is coated with a colloidal gold-labeled noraconazole monoclonal antibody-nano silver composite probe. The nitrocellulose membrane 50 is provided with a detection line 51 and a control line 52.

[0023] The detection line 51 is coated with a novogene hapten-bovine serum albumin conjugate, and the control line 52 is coated with goat anti-mouse IgG antibody; the sample pad 30 is soaked in a composite pretreatment solution containing Tris-HCl buffer, Tween-20, bovine serum albumin and disodium EDTA.

[0024] The top left side of the casing 10 has a sample application hole 11, which is located directly above the sample pad 30; the top right side of the casing 10 has an observation window 12, which is located directly above the nitrocellulose membrane 50.

[0025] A method for preparing a nocoagulant detection card includes the following specific steps: Preparation of S1, Novogene monoclonal antibody: S11, Hapten Synthesis: Using nifedipine as a raw material, succinic anhydride was added as a modifier. The reaction was carried out in pyridine solvent at 42.5℃ for 3.5 hours. After the reaction was completed, deionized water was added for dilution, and the pH was adjusted to 2.5 with hydrochloric acid to precipitate the precipitate. The precipitate was washed three times with ether and purified by silica gel column chromatography. The target component was collected and vacuum dried to obtain the nifedipine hapten. S12, Immunogen Preparation: Take the nifedipine hapten prepared in S11, add N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and activate at room temperature for 25 minutes to obtain the hapten activation solution; dissolve bovine serum albumin (BSA) in PBS buffer (pH 7.4), and slowly add it dropwise to the hapten activation solution, and stir at 4°C for 14 hours; after the reaction, dialyze with PBS buffer for 3 days, changing the dialysate 3 times a day to remove unbound hapten, and obtain the nifedipine hapten-BSA conjugate (immunogen). S13, Animal Immunization: Seven-week-old female BALB / c mice were selected. The novacrine hapten-BSA conjugate prepared in S12 was mixed with an equal volume of Freund's complete adjuvant and emulsified, and then injected intraperitoneally for immunization. The immunization dose was 50 μg / mouse. After the first immunization, booster immunizations were performed every two weeks. For booster immunizations, the novacrine hapten-BSA conjugate was mixed with an equal volume of Freund's incomplete adjuvant and emulsified, and the immunization dose was 30 μg / mouse. A total of three booster immunizations were performed. Seven days after the last immunization, tail vein blood was collected from the mice, and serum antibody titers were detected by indirect ELISA. Mice with antibody titers higher than 1:100000 were selected as donors. S14, Hybridoma Cell Preparation and Screening: Spleens from mice with qualified antibody titers in S13 were collected, ground, and splenocytes were obtained. Splenocytes were mixed with myeloma cells (SP2 / 0) at a ratio of 5:1, and polyethylene glycol (PEG4000) was added for fusion. After fusion, the cells were seeded into 96-well cell culture plates containing HAT selective medium and incubated at 37°C with 5% [temperature missing]. The cells were cultured in an incubator. After 8 days of culture, positive wells were screened by indirect ELISA. The cells in the positive wells were then subjected to three limiting dilution clonal culture to finally obtain hybridoma cell lines that could stably secrete norostatin monoclonal antibodies. S15, Monoclonal Antibody Purification: The hybridoma cell line obtained in S14 was inoculated into the peritoneal cavity of mice to induce ascites; after collecting the ascites, it was purified using a Protein G affinity chromatography column. The chromatography column was first equilibrated with equilibration buffer, then the ascites was loaded, the target antibody was eluted with elution buffer, the elution peak was collected and dialyzed to desalt, and high-purity nifedipine monoclonal antibody was obtained. S2, Preparation of colloidal gold-labeled novolucretin monoclonal antibody-silver nanoparticle composite probe: S21, Preparation of colloidal gold particles: Using the sodium citrate reduction method, 100 mL of 0.01% chloroauric acid solution was placed in a three-necked flask and heated to boiling. 10 mL of 1% sodium citrate solution was quickly added and the mixture was boiled for 17.5 minutes. The solution gradually changed from pale yellow to wine red. Heating was stopped and the solution was cooled to room temperature to obtain a colloidal gold solution with a particle size of 25 nm. S22, Silver-modified colloidal gold particles: Take the colloidal gold solution prepared in S21, and use 0.1 mol / L... The pH of the solution was adjusted to 8.75, 10 mL of 0.05% silver nitrate solution was added, and after stirring evenly, 5 mL of 0.1% sodium borohydride solution was slowly added dropwise. The reaction was carried out at room temperature in the dark with stirring for 1 hour to obtain colloidal gold nanoparticles modified with silver nanoparticles. After the reaction was completed, the mixture was centrifuged at 10000 r / min for 20 minutes, the supernatant was discarded, and the precipitate was resuspended in PBS buffer (pH 7.4) containing 0.02% sodium azide to obtain a solution of silver nanoparticle-colloidal gold composite particles. S23, Composite Probe Labeling: Take the nano-silver-colloidal gold composite particle solution prepared in S22, add the purified nifedipine monoclonal antibody from S15, the antibody addition amount is 50 μg / mL, and stir at room temperature in the dark for 1 hour; add bovine serum albumin (BSA) to the final concentration of 1%, continue stirring for 30 minutes to block the unbound active sites; then centrifuge at 12000 r / min for 30 minutes, discard the supernatant, resuspend the precipitate in PBS buffer (pH 7.4) containing 0.5% Tween-20, 1% BSA and 0.02% sodium azide, and adjust the probe concentration to OD520nm=1.5 to obtain the colloidal gold-labeled nifedipine monoclonal antibody-nano-silver composite probe; S24, Crosslinking and Curing Modification of Composite Probe: Take the composite probe solution prepared in S23, add glutaraldehyde crosslinking agent to a final concentration of 0.2%, and stir at room temperature in the dark for 40 minutes; utilize the bifunctional characteristics of glutaraldehyde to initially activate the active sites on the probe surface; add 0.5% polyethyleneimine (PEI) to the reaction solution and continue stirring for 20 minutes to form a stable structure of probe-PEI-crosslinking agent, enhancing the adsorption force between the probe and the binding pad substrate; add glycine to a final concentration of 1% and stir for 15 minutes to block unreacted crosslinking sites and avoid subsequent nonspecific binding; centrifuge at 12000 r / min for 30 minutes, discard the supernatant, and resuspend the precipitate with the original probe resuspending buffer to obtain the crosslinked and cured composite probe; S3, Preparation of core components of the detection card: S31, Sample pad 30 pretreatment: Take a glass fiber membrane as the sample pad substrate and immerse it in a composite pretreatment solution for 30 minutes; the composite pretreatment solution consists of 20 mmol / L Tris-HCl buffer (pH 8.0), 0.5% Tween-20, 2% bovine serum albumin and 0.1% disodium EDTA; after immersion, place it in a 37℃ oven to dry for 2 hours, and set aside for later use; S32, Hydrophilic-hydrophobic gradient modification of nitrocellulose membrane 50: A gradient modification solution was prepared by adding Tris-HCl buffer (pH 8.0) containing 0.1%, 0.3%, and 0.5% Tween-80 to form a hydrophilic gradient from low to high. The nitrocellulose membrane 50 was laid flat on the modification platform. Using the dip-pull method, the end of the nitrocellulose membrane 50 near the binding pad 40 was first immersed in a 0.5% Tween-80 solution (strong hydrophilicity, facilitating probe migration), and the end near the absorbent pad 60 was immersed in a 0.1% Tween-80 solution (weak hydrophilicity, preventing band diffusion). The middle region was transitioned by a gradient in the pulling speed (2 mm / s → 1 mm / s). The modified nitrocellulose membrane 50 was then placed in a 37°C oven for 2 hours to solidify the gradient structure. S33, Preparation of bonding pad 40: A polyester film was used as the bonding pad substrate. The cross-linked and cured composite probe prepared in S24 was uniformly sprayed onto the polyester film at a spraying amount of 8 μL / cm². After spraying, the film was placed in a vacuum drying oven at 37℃ for 1 hour. After drying, it was sealed and stored in a dry environment for later use. S34, Nitrocellulose Membrane 50 Treatment: Take the S32 modified nitrocellulose membrane 50 and use a membrane scrubbing instrument to divide the nitrocellulose membrane 50 into a detection line 51 (T line) and a control line 52 (C line) with the concentration of 1 mg / mL of the nitrocellulose hapten-bovine serum albumin conjugate and goat anti-mouse IgG antibody (2 mg / mL). The distance between the detection line 51 and the control line 52 is 5 mm, and the scrubbing speed is 3 cm / s. After scrubbing, place the membrane in a 37℃ oven to dry for 3 hours for later use. S35, Pre-treatment of absorbent pad 60: Take absorbent paper as the substrate of absorbent pad, cut it to the size that matches the PVC base plate 20, place it in a 120℃ oven to dry for 30 minutes to remove moisture, and set aside. S4: Assembly of the detection card: S41, Component overlap: Take a PVC base plate 20 and, in the order of sample pad 30, binding pad 40, nitrocellulose membrane 50, and absorbent pad 60, overlap and fix the sample pad 30 (pretreated in S31), binding pad 40 (prepared in S33), nitrocellulose membrane 50 (treated in S34), and absorbent pad 60 (pretreated in S35) onto the PVC base plate 20 in sequence; wherein, the right end of the binding pad overlaps the left end of the NC membrane (overlap length is 2mm), the right end of the NC membrane overlaps the left end of the absorbent pad (overlap length is 2mm), the left end of the sample pad is aligned with the left end of the PVC base plate, and the right end of the absorbent pad is aligned with the right end of the PVC base plate; S42, cassette assembly: Install the overall structure completed in S41 into the cassette 10, ensuring that the sample pad 30 corresponds to the sample application hole 11 of the cassette 10, and that the detection line 51 and quality control line 52 of the nitrocellulose membrane 50 correspond to the observation window 12 of the cassette 10; after assembly, use a tablet press to compact the cassette, ensuring that all components are firmly fixed. Example 3:

[0026] This invention provides a norepinephrine detection card; please refer to [link / reference]. Figures 1-3 The device includes a housing 10, with a PVC base plate 20 fixedly installed at the bottom of the inner cavity of the housing 10. The top of the PVC base plate 20 is sequentially overlapped and fixed with a sample pad 30, a conjugate pad 40, a nitrocellulose membrane 50, and an absorbent pad 60 from left to right. The conjugate pad 40 is coated with a colloidal gold-labeled noraconazole monoclonal antibody-nano silver composite probe. The nitrocellulose membrane 50 is provided with a detection line 51 and a control line 52.

[0027] The detection line 51 is coated with a novogene hapten-bovine serum albumin conjugate, and the control line 52 is coated with goat anti-mouse IgG antibody; the sample pad 30 is soaked in a composite pretreatment solution containing Tris-HCl buffer, Tween-20, bovine serum albumin and disodium EDTA.

[0028] The top left side of the casing 10 has a sample application hole 11, which is located directly above the sample pad 30; the top right side of the casing 10 has an observation window 12, which is located directly above the nitrocellulose membrane 50.

[0029] A method for preparing a nocoagulant detection card includes the following specific steps: Preparation of S1, Novogene monoclonal antibody: S11, Hapten Synthesis: Using nifedipine as a raw material, succinic anhydride was added as a modifier and reacted at 45°C for 4 hours in pyridine solvent. After the reaction, deionized water was added for dilution, and the pH was adjusted to 3.0 with hydrochloric acid to precipitate the precipitate. The precipitate was washed three times with ether and purified by silica gel column chromatography. The target component was collected and vacuum dried to obtain the nifedipine hapten. S12, Immunogen Preparation: Take the nifedipine hapten prepared in S11, add N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and activate at room temperature for 30 minutes to obtain the hapten activation solution; dissolve bovine serum albumin (BSA) in PBS buffer (pH 7.4), and slowly add it dropwise to the hapten activation solution, and stir at 4°C for 16 hours; after the reaction, dialyze with PBS buffer for 3 days, changing the dialysate 3 times a day to remove unbound hapten, and obtain the nifedipine hapten-BSA conjugate (immunogen). S13, Animal Immunization: Eight-week-old female BALB / c mice were selected. The novacrine hapten-BSA conjugate prepared in S12 was mixed with an equal volume of Freund's complete adjuvant and emulsified, and then injected intraperitoneally for immunization. The immunization dose was 50 μg / mouse. After the first immunization, booster immunizations were performed every 2 weeks. For booster immunizations, the novacrine hapten-BSA conjugate was mixed with an equal volume of Freund's incomplete adjuvant and emulsified, and the immunization dose was 30 μg / mouse. A total of 3 booster immunizations were performed. Seven days after the last immunization, tail vein blood was collected from the mice, and serum antibody titers were detected by indirect ELISA. Mice with antibody titers higher than 1:100000 were selected as donors. S14, Hybridoma Cell Preparation and Screening: Spleens from mice with qualified antibody titers in S13 were collected, ground, and splenocytes were obtained. Splenocytes were mixed with myeloma cells (SP2 / 0) at a ratio of 5:1, and polyethylene glycol (PEG4000) was added for fusion. After fusion, the cells were seeded into 96-well cell culture plates containing HAT selective medium and incubated at 37°C with 5% [temperature missing]. The cells were cultured in an incubator. After 10 days of culture, positive wells were screened by indirect ELISA. The cells in the positive wells were then subjected to three limiting dilution clonal culture to finally obtain hybridoma cell lines that could stably secrete norostatin monoclonal antibodies. S15, Monoclonal Antibody Purification: The hybridoma cell line obtained in S14 was inoculated into the peritoneal cavity of mice to induce ascites; after collecting the ascites, it was purified using a Protein G affinity chromatography column. The chromatography column was first equilibrated with equilibration buffer, then the ascites was loaded, the target antibody was eluted with elution buffer, the elution peak was collected and dialyzed to desalt, and high-purity nifedipine monoclonal antibody was obtained. S2, Preparation of colloidal gold-labeled novolucretin monoclonal antibody-silver nanoparticle composite probe: S21, Preparation of colloidal gold particles: Using the sodium citrate reduction method, 100 mL of 0.01% chloroauric acid solution was placed in a three-necked flask and heated to boiling. 10 mL of 1% sodium citrate solution was quickly added and the mixture was boiled for another 20 minutes. The solution gradually changed from pale yellow to wine red. Heating was stopped and the mixture was cooled to room temperature to obtain a colloidal gold solution with a particle size of 30 nm. S22, Silver-modified colloidal gold particles: Take the colloidal gold solution prepared in S21, and use 0.1 mol / L... The pH of the solution was adjusted to 9.0, 10 mL of 0.05% silver nitrate solution was added, and after stirring, 5 mL of 0.1% sodium borohydride solution was slowly added dropwise. The reaction was carried out at room temperature in the dark with stirring for 1 hour to obtain colloidal gold nanoparticles modified with silver nanoparticles. After the reaction was completed, the mixture was centrifuged at 10000 r / min for 20 minutes, the supernatant was discarded, and the precipitate was resuspended in PBS buffer (pH 7.4) containing 0.02% sodium azide to obtain a solution of silver nanoparticle-colloidal gold composite particles. S23, Composite Probe Labeling: Take the nano-silver-colloidal gold composite particle solution prepared in S22, add the purified nifedipine monoclonal antibody from S15, the antibody addition amount is 50 μg / mL, and stir at room temperature in the dark for 1 hour; add bovine serum albumin (BSA) to the final concentration of 1%, continue stirring for 30 minutes to block the unbound active sites; then centrifuge at 12000 r / min for 30 minutes, discard the supernatant, resuspend the precipitate in PBS buffer (pH 7.4) containing 0.5% Tween-20, 1% BSA and 0.02% sodium azide, and adjust the probe concentration to OD520nm=1.5 to obtain the colloidal gold-labeled nifedipine monoclonal antibody-nano-silver composite probe; S24, Crosslinking and Curing Modification of Composite Probe: Take the composite probe solution prepared in S23, add glutaraldehyde crosslinking agent to a final concentration of 0.2%, and stir at room temperature in the dark for 40 minutes; utilize the bifunctional characteristics of glutaraldehyde to initially activate the active sites on the probe surface; add 0.5% polyethyleneimine (PEI) to the reaction solution and continue stirring for 20 minutes to form a stable structure of probe-PEI-crosslinking agent, enhancing the adsorption force between the probe and the binding pad substrate; add glycine to a final concentration of 1% and stir for 15 minutes to block unreacted crosslinking sites and avoid subsequent nonspecific binding; centrifuge at 12000 r / min for 30 minutes, discard the supernatant, and resuspend the precipitate with the original probe resuspending buffer to obtain the crosslinked and cured composite probe; S3, Preparation of core components of the detection card: S31, Sample pad 30 pretreatment: Take a glass fiber membrane as the sample pad substrate and immerse it in a composite pretreatment solution for 30 minutes; the composite pretreatment solution consists of 20 mmol / L Tris-HCl buffer (pH 8.0), 0.5% Tween-20, 2% bovine serum albumin and 0.1% disodium EDTA; after immersion, place it in a 37℃ oven to dry for 2 hours, and set aside for later use; S32, Hydrophilic-hydrophobic gradient modification of nitrocellulose membrane 50: A gradient modification solution was prepared by adding Tris-HCl buffer (pH 8.0) containing 0.1%, 0.3%, and 0.5% Tween-80 to form a hydrophilic gradient from low to high. The nitrocellulose membrane 50 was laid flat on the modification platform. Using the dip-pull method, the end of the nitrocellulose membrane 50 near the binding pad 40 was first immersed in a 0.5% Tween-80 solution (strong hydrophilicity, facilitating probe migration), and the end near the absorbent pad 60 was immersed in a 0.1% Tween-80 solution (weak hydrophilicity, preventing band diffusion). The middle region was transitioned by a gradient in the pulling speed (2 mm / s → 1 mm / s). The modified nitrocellulose membrane 50 was then placed in a 37°C oven for 2 hours to solidify the gradient structure. S33, Preparation of bonding pad 40: A polyester film was used as the bonding pad substrate. The cross-linked and cured composite probe prepared in S24 was uniformly sprayed onto the polyester film at a spraying amount of 8 μL / cm². After spraying, the film was placed in a vacuum drying oven at 37℃ for 1 hour. After drying, it was sealed and stored in a dry environment for later use. S34, Nitrocellulose Membrane 50 Treatment: Take the S32 modified nitrocellulose membrane 50 and use a membrane scrubbing instrument to divide the nitrocellulose membrane 50 into a detection line 51 (T line) and a control line 52 (C line) with the concentration of 1 mg / mL of the nitrocellulose hapten-bovine serum albumin conjugate and goat anti-mouse IgG antibody (2 mg / mL). The distance between the detection line 51 and the control line 52 is 5 mm, and the scrubbing speed is 3 cm / s. After scrubbing, place the membrane in a 37℃ oven to dry for 3 hours for later use. S35, Pre-treatment of absorbent pad 60: Take absorbent paper as the substrate of absorbent pad, cut it to the size that matches the PVC base plate 20, place it in a 120℃ oven to dry for 30 minutes to remove moisture, and set aside. S4: Assembly of the detection card: S41, Component overlap: Take a PVC base plate 20 and, in the order of sample pad 30, binding pad 40, nitrocellulose membrane 50, and absorbent pad 60, overlap and fix the sample pad 30 (pretreated in S31), binding pad 40 (prepared in S33), nitrocellulose membrane 50 (treated in S34), and absorbent pad 60 (pretreated in S35) onto the PVC base plate 20 in sequence; wherein, the right end of the binding pad overlaps the left end of the NC membrane (overlap length is 2mm), the right end of the NC membrane overlaps the left end of the absorbent pad (overlap length is 2mm), the left end of the sample pad is aligned with the left end of the PVC base plate, and the right end of the absorbent pad is aligned with the right end of the PVC base plate; S42, cassette assembly: Install the overall structure completed in S41 into the cassette 10, ensuring that the sample pad 30 corresponds to the sample application hole 11 of the cassette 10, and that the detection line 51 and quality control line 52 of the nitrocellulose membrane 50 correspond to the observation window 12 of the cassette 10; after assembly, use a tablet press to compact the cassette, ensuring that all components are firmly fixed.

[0030] The following data were obtained by comparing the Nocogenin test cards prepared in Examples 1-3 above: Example 1 Example 2 Example 3 Limit of detection 2ng / mL 1ng / mL 1ng / mL Performance degradation rate after 12 months of storage at room temperature ＜9% ＜8% ＜10% Liquid migration velocity deviation ＜5% ＜4% ＜5% As shown in the table above, the Nocogenin test cards prepared in Examples 1-3 all showed good performance in terms of the lowest detection limit, the performance degradation rate after 12 months of storage at room temperature, and the deviation of liquid migration speed. After use, Example 2 showed the best results.

[0031] Although the present invention has been described above with reference to embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of the invention. In particular, as long as there is no structural conflict, the features in the disclosed embodiments can be combined with each other in any manner. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A novel angiotensin II test card, comprising a card housing (10), wherein a PVC base plate (20) is fixedly installed at the bottom of the inner cavity of the card housing (10), characterized in that, The top of the PVC base plate (20) is sequentially connected from left to right to fix the sample pad (30), the conjugate pad (40), the nitrocellulose membrane (50) and the absorbent pad (60). The conjugate pad (40) is coated with colloidal gold-labeled nocoagulant monoclonal antibody-nano silver composite probe. The nitrocellulose membrane (50) is provided with a detection line (51) and a quality control line (52).

2. The noradrenaline test card according to claim 1, characterized in that, The detection line (51) is coated with a novogamine hapten-bovine serum albumin conjugate, and the control line (52) is coated with goat anti-mouse IgG antibody.

3. The noradrenaline detection card according to claim 1, characterized in that, The sample pad (30) is soaked in a composite pretreatment solution containing Tris-HCl buffer, Tween-20, bovine serum albumin and disodium EDTA.

4. The noradrenaline test card according to claim 1, characterized in that, The top left side of the casing (10) has a sample loading hole (11), and the sample loading hole (11) is located directly above the sample pad (30).

5. The Novogene test card according to claim 1, characterized in that, The top right side of the casing (10) is provided with an observation window (12), and the observation window (12) is located directly above the nitrocellulose membrane (50).

6. A method for preparing a norepinephrine detection card, characterized in that, The specific steps are as follows: Preparation of S1, Novogene monoclonal antibody: S11, Hapten Synthesis: Using nifedipine as a raw material, succinic anhydride was added as a modifier and the reaction was carried out at a constant temperature in a pyridine solvent. After the reaction was completed, deionized water was added for dilution, and the pH was adjusted to 2.0-3.0 with hydrochloric acid to precipitate the precipitate. The precipitate was washed three times with diethyl ether and then purified by silica gel column chromatography. The target component was collected and vacuum dried to obtain the nifedipine hapten. S12, Immunogen Preparation: Take the nifedipine hapten prepared in S11, add N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and activate at room temperature for 20-30 minutes to obtain the hapten activation solution; dissolve bovine serum albumin in PBS buffer and slowly add it dropwise to the hapten activation solution, and stir the reaction; after the reaction is completed, dialyze with PBS buffer for 3 days, changing the dialysate 3 times a day to remove unbound hapten, and obtain the nifedipine hapten-BSA conjugate; S13, Animal Immunization: 6-8 week old female BALB / c mice were selected. The novacrine hapten-BSA conjugate prepared in S12 was mixed with an equal volume of Freund's complete adjuvant and emulsified, and then injected intraperitoneally. The immunization dose was 50 μg / mouse. After the first immunization, booster immunizations were performed every 2 weeks. For booster immunizations, the novacrine hapten-BSA conjugate was mixed with an equal volume of Freund's incomplete adjuvant and emulsified, and the immunization dose was 30 μg / mouse. A total of 3 booster immunizations were performed. Seven days after the last immunization, tail vein blood was collected from the mice, and serum antibody titers were detected by indirect ELISA. Mice with antibody titers higher than 1:100000 were selected as donors. S14, Hybridoma Cell Preparation and Screening: Spleens from mice with qualified antibody titers from S13 were collected, ground, and splenocytes were obtained. Splenocytes were mixed with myeloma cells at a ratio of 5:1, and polyethylene glycol was added for fusion. After fusion, the cells were seeded into 96-well cell culture plates containing HAT selective medium and incubated at 37°C with 5% [temperature missing]. Cultured in an incubator; after 7-10 days of culture, positive wells were screened by indirect ELISA, and the cells in the positive wells were subjected to three limiting dilution clonal culture to finally obtain hybridoma cell lines that can stably secrete norostatin monoclonal antibodies; S15, Monoclonal Antibody Purification: The hybridoma cell line obtained in S14 was inoculated into the peritoneal cavity of mice to induce ascites; after collecting the ascites, it was purified using a Protein G affinity chromatography column. The chromatography column was first equilibrated with equilibration buffer, then the ascites was loaded, the target antibody was eluted with elution buffer, the elution peak was collected and dialyzed to desalt, and high-purity nifedipine monoclonal antibody was obtained. S2, Preparation of colloidal gold-labeled novolucretin monoclonal antibody-silver nanoparticle composite probe: S21, Preparation of colloidal gold particles: Using the sodium citrate reduction method, 100 mL of 0.01% chloroauric acid solution was placed in a three-necked flask and heated to boiling. 10 mL of 1% sodium citrate solution was quickly added and the mixture was boiled for 15-20 minutes. The solution gradually changed from pale yellow to wine red. Heating was stopped and the solution was cooled to room temperature to obtain a colloidal gold solution with a particle size of 20-30 nm. S22, Silver-modified colloidal gold particles: Take the colloidal gold solution prepared in S21, and use 0.1 mol / L... The pH of the solution was adjusted to 8.5-9.0, 10 mL of 0.05% silver nitrate solution was added, and after stirring evenly, 5 mL of 0.1% sodium borohydride solution was slowly added dropwise. The reaction was carried out at room temperature in the dark for 1 hour to obtain colloidal gold nanoparticles modified with silver nanoparticles. After the reaction was completed, the supernatant was discarded after centrifugation, and the precipitate was resuspended in PBS buffer containing 0.02% sodium azide to obtain a solution of silver nanoparticle-colloidal gold composite particles. S23, Composite Probe Labeling: Take the nano-silver-colloidal gold composite particle solution prepared in S22, add the purified nifedipine monoclonal antibody from S15, the antibody addition amount is 50 μg / mL, and stir at room temperature in the dark for 1 hour; add bovine serum albumin to the final concentration of 1%, continue stirring for 30 minutes to block the unbound active sites; then centrifuge at 12000 r / min for 30 minutes, discard the supernatant, resuspend the precipitate in PBS buffer containing 0.5% Tween-20, 1% BSA and 0.02% sodium azide, adjust the probe concentration to OD520nm=1.5, and obtain the colloidal gold-labeled nifedipine monoclonal antibody-nano-silver composite probe; S24, Crosslinking and Curing Modification of Composite Probe: Take the composite probe solution prepared in S23, add glutaraldehyde crosslinking agent to a final concentration of 0.2%, and stir at room temperature in the dark for 40 minutes; utilize the bifunctional characteristics of glutaraldehyde to initially activate the active sites on the probe surface; add 0.5% polyethyleneimine to the reaction solution and continue stirring for 20 minutes to form a stable structure of probe-PEI-crosslinking agent, enhancing the adsorption force between the probe and the binding pad substrate; add glycine to a final concentration of 1% and stir for 15 minutes to block unreacted crosslinking sites and avoid subsequent nonspecific binding; centrifuge at 12000 r / min for 30 minutes, discard the supernatant, and resuspend the precipitate with the original probe resuspending buffer to obtain the crosslinked and cured composite probe; S3, Preparation of core components of the detection card: S31, Sample pad (30) pretreatment: Take glass fiber membrane as the sample pad substrate, immerse it in composite pretreatment solution for 30 minutes; after immersion, place it in an oven to dry for later use; S32, Hydrophilic-hydrophobic gradient modification of nitrocellulose membrane (50): Prepare gradient modification solutions, and prepare Tris-HCl buffer solutions containing 0.1%, 0.3%, and 0.5% Tween-80 respectively to form a hydrophilic gradient solution from low to high; lay the nitrocellulose membrane (50) flat on the modification platform, and use the soak-lift method, first soak the end of the nitrocellulose membrane (50) near the binding pad (40) in 0.5% Tween-80 solution, and the end near the absorbent pad (60) in 0.1% Tween-80 solution, and the middle area is transitioned by the lifting speed gradient; place the modified nitrocellulose membrane (50) in an oven for drying to solidify the gradient structure; S33, preparation of bonding pad (40): take polyester film as bonding pad substrate, and uniformly spray the cross-linked and cured modified composite probe prepared in S24 onto the polyester film. The spraying amount is 8μL / cm². After spraying, place it in a vacuum drying oven for drying. After drying, seal and store it in a dry environment for later use. S34, Nitrocellulose membrane (50) treatment: Take the nitrocellulose membrane (50) modified by S32, and use a membrane scrubbing instrument to divide the nitrocellulose hapten-bovine serum albumin conjugate and goat anti-mouse IgG antibody into a detection line (51) and a control line (52) respectively. The distance between the detection line (51) and the control line (52) is 5 mm, and the scrubbing speed is 3 cm / s. After scrubbing, place it in an oven to dry for later use. S35, Pretreatment of absorbent pad (60): Take absorbent paper as the substrate of absorbent pad, cut it to the size that matches the PVC base plate (20), place it in a 120℃ oven to dry for 30 minutes to remove moisture, and set aside. S4: Assembly of the detection card: S41, Component overlap: Take the PVC base plate (20) and, in the order of sample pad (30), conjugate pad (40), nitrocellulose membrane (50) and absorbent pad (60), overlap and fix the sample pad (30) pretreated in S31, the conjugate pad (40) prepared in S33, the nitrocellulose membrane (50) treated in S34 and the absorbent pad (60) pretreated in S35 onto the PVC base plate (20) in sequence. S42, cassette assembly: Install the overall structure completed in S41 into the cassette (10), ensuring that the sample pad (30) corresponds to the sample loading hole (11) of the cassette (10), and that the detection line (51) and quality control line (52) of the nitrocellulose membrane (50) correspond to the observation window (12) of the cassette (10); after assembly, use a tablet press to compact the cassette and ensure that each component is firmly fixed.

7. The method for preparing a norepinephrine detection card according to claim 6, characterized in that, In step S11, the reaction temperature in the pyridine solvent is set to 40-45°C, and the reaction time is set to 3-4 hours; in step S12, the reaction temperature in the hapten activation solution is set to 4°C, and the reaction time is set to 12-16 hours.

8. The method for preparing a noradrenaline detection card according to claim 6, characterized in that, In step S22, after the reaction is complete, the centrifugation speed is set to 10000 r / min and the centrifugation time is set to 20 minutes.

9. The method for preparing a noradrenaline detection card according to claim 6, characterized in that, In step S31, the raw materials of the composite pretreatment solution, calculated by percentage, include: 20 mmol / L Tris-HCl buffer, 0.5% Tween-20, 2% bovine serum albumin, and 0.1% disodium EDTA. The drying temperature after soaking is set to 37°C, and the drying time is set to 2 hours.

10. The method for preparing a noradrenaline detection card according to claim 6, characterized in that, In step S32, the drying temperature of the oven is set to 37°C and the drying time is set to 2 hours; in step S33, the drying temperature of the vacuum drying oven is set to 37°C and the drying time is set to 1 hour; in step S34, the drying temperature of the oven is set to 37°C and the drying time is set to 3 hours.

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