Preparation method and application of a time-resolved fluorescence immunochromatographic test strip for detecting difenoconazole in strawberry
By preparing difenoconazole hapten and antibody-time-resolved fluorescent microspheres, the sensitivity and stability issues of difenoconazole detection in strawberries in existing technologies have been solved, achieving high sensitivity and specificity in detection, and the test strips have a long shelf life.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUIZHOU ACADEMY OF TESTING & ANALYSIS
- Filing Date
- 2023-05-12
- Publication Date
- 2026-06-09
AI Technical Summary
Existing technologies for detecting difenoconazole in strawberries have low sensitivity, poor specificity, and poor stability, making them unsuitable for long-term storage.
A time-resolved fluorescent immunochromatographic test strip was prepared by employing methods including the preparation of difenoconazole hapten, preparation of difenoconazole antibody, preparation of difenoconazole antibody-time-resolved fluorescent microspheres, preparation of fluorescent microsphere pads, and test strip assembly. This process includes the synthesis of difenoconazole hapten, preparation of antibody, and preparation of microspheres. Combined with specific concentration and temperature treatments, the high sensitivity and specificity of the test strip are ensured.
It achieves highly sensitive detection of difenoconazole in strawberries, with specific identification, low detection limit, good stability, and a shelf life of over 12 months.
Smart Images
Figure CN116466077B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological detection technology, specifically a method for preparing and applying a time-resolved fluorescent immunochromatographic test strip for detecting difenoconazole in strawberries. Background Technology
[0002] Strawberry powdery mildew is caused by the fungus *Monophyllum hygroscopicum*, a member of the Ascomycota. It is a significant disease in strawberry production, primarily affecting leaves and fruits. In severe cases, the disease incidence rate can exceed 45% for leaves and 50% for fruits. Difenoconazole fungicide is widely used for controlling strawberry powdery mildew due to its high efficiency, broad spectrum, low toxicity, long residual effect, and good systemic conductivity. However, improper use by some farmers has led to excessive levels of difenoconazole residues in strawberry fruits. To improve food safety, it is necessary to test for difenoconazole residues in strawberries to prevent strawberries with excessive levels of difenoconazole from entering the market.
[0003] Chinese patent discloses a test strip and method for detecting difenoconazole (authorization announcement number CN112067814A). This patented technology includes a sample absorption pad, a conjugate release pad, a reaction membrane, an absorbent pad, and a base plate. The reaction membrane has a detection line coated with a difenoconazole hapten-carrier protein conjugate and a control line coated with goat anti-mouse anti-antibody. The conjugate release pad is coated with a difenoconazole monoclonal antibody-colloidal gold label. It has advantages such as simple operation, high sensitivity, fast detection speed, low cost, and suitability for large-scale sample screening. However, it has poor specificity for difenoconazole and cannot be stored for long periods, exhibiting poor stability. Summary of the Invention
[0004] The purpose of this invention is to provide a method for preparing and applying a time-resolved fluorescent immunochromatographic test strip for detecting difenoconazole in strawberries, in order to solve the problems mentioned in the background art.
[0005] To achieve the above objectives, the present invention provides the following technical solution:
[0006] A method for preparing a time-resolved fluorescence immunochromatographic test strip for detecting difenoconazole in strawberries includes the following steps:
[0007] S1. Preparation of difenoconazole hapten: The difenoconazole hapten was prepared by using 4-(3-methyl-4-chlorophenyl)oxyacetophenone, 1,2-propanediol, p-toluenesulfonic acid, bromine, DMF, potassium triazole salt, potassium permanganate, and thionyl chloride, and then dissolving, distilling, crystallizing, and purifying to obtain the difenoconazole hapten.
[0008] S2. Preparation of difenoconazole antibody: The difenoconazole hapten was conjugated with bovine serum albumin to obtain the difenoconazole immunogen; mice were then immunized with the difenoconazole immunogen, and then the mouse spleen cells and myeloma cells were fused and screened to obtain the difenoconazole antibody;
[0009] S3. Preparation of difenoconazole antibody-time-resolved fluorescent microspheres: N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added to 2-morpholine ethanesulfonic acid to prepare a mixed solution; then rare earth fluorescent complex microspheres and difenoconazole antibody were added to obtain difenoconazole antibody-time-resolved fluorescent microspheres.
[0010] S4. Preparation of fluorescent microsphere pads: The polyester fiber layer was immersed in phosphate buffer containing bovine serum albumin. After immersion, it was dried at low temperature. Then, the difenoconazole antibody-time-resolved fluorescent microspheres were uniformly sprayed onto the polyester fiber layer and dried at low temperature again to obtain the fluorescent microsphere pads.
[0011] S5. Preparation of reaction membrane: The difenoconazole hapten is coupled with ovalbumin to obtain the difenoconazole coated antigen, and then the difenoconazole coated antigen is coated onto a nitrocellulose membrane to form a detection line; the goat anti-mouse IgG antibody is coated onto a nitrocellulose membrane to form a quality control line, thus obtaining the reaction membrane;
[0012] S6. Test strip assembly: Attach the absorbent pad, reaction membrane, fluorescent microsphere pad, and sample pad to the base plate in sequence, and then cut them into test strips 3-4 mm wide to obtain time-resolved fluorescence immunochromatographic test strips. Place them in a foil bag, add desiccant, and seal for storage.
[0013] As a further embodiment of the present invention: the preparation method of the difenoconazole hapten in step S1 includes the following steps:
[0014] S11. Dissolve 4-(3-methyl-4-chlorophenyl)oxyacetophenone in toluene in a round-bottom flask, add 1,2-propanediol and p-toluenesulfonic acid, raise the temperature to 120℃ and react. Use a water separator to separate the water produced in the reaction. When no more water is produced, the reaction is complete. Cool the reaction solution to room temperature, separate the lower layer of the reaction solution with a separatory funnel, wash it three times with saturated saline, and then wash it twice with distilled water to obtain a pale yellow oily liquid, which is compound A.
[0015] S12. Dissolve compound A in cyclohexane, and add one-third of the measured amount of bromine dropwise while stirring at room temperature. After the addition is complete, wait for the red color of the reaction solution to disappear, and then lower the temperature to 20°C. Continue to add dropwise. After the addition is complete, heat the reaction solution in an oil bath under reflux and detect the reaction by TLC. After the reaction is complete, cool the reaction solution to room temperature and remove the solvent by vacuum distillation to obtain a yellowish-brown oily liquid, which is compound B.
[0016] S13. Compound B was dissolved in DMF, and potassium triazole salt was added. The reaction was carried out at 150°C, and the reaction was detected by TLC. The reaction was completed after 8 hours. The reaction solution was distilled under reduced pressure to recover DMF. Then water and toluene were added, and the crude product was extracted with a separatory funnel. The product was dried with anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure to obtain a yellowish-brown solid. The solid was recrystallized from ethanol to obtain pale yellow needle-like crystals, which were compound C.
[0017] S14. Dissolve compound C in acetonitrile, add an appropriate amount of water to make the solution just clear, add potassium permanganate in portions under ice bath, remove the ice bath after the addition is complete, react at room temperature for 4 hours, filter the reaction solution, wash the filter cake twice with acetonitrile, take the filtrate, distill under reduced pressure to remove acetonitrile, dissolve the residue in ethyl acetate, dry sample loading and column purification, collect the main UV spot components, and after desolvation by reduced pressure distillation, obtain a white solid, which is compound D;
[0018] S15. Compound D was dissolved in thionyl chloride, and 3 drops of DMF were added. The mixture was reacted at 116°C for 4 hours. After the reaction was complete, the reaction solution was distilled under reduced pressure, and the remaining small amount of thionyl chloride was removed with n-hexane to obtain compound E. Compound E was dissolved in anhydrous tetrahydrofuran and cooled to 0°C for later use. Separately, 4-aminobutyric acid was dissolved in 1N NaOH and cooled to 0°C. The tetrahydrofuran solution of compound E was added in 5 batches to the sodium hydroxide solution of 4-aminobutyric acid, with each batch at least 10 minutes apart. After the addition was complete, the reaction was carried out in an ice bath. After about 3 hours, the reaction was complete. The reaction solution was distilled under reduced pressure to remove the tetrahydrofuran. An appropriate amount of water was added to the residue, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness. The product was purified by silica gel column chromatography to obtain a pale yellow solid, which is the hapten of difenoconazole. The synthetic route of the difenoconazole hapten is as follows:
[0019]
[0020] As a further embodiment of the present invention: the preparation method in step S3 includes the following steps:
[0021] S31. Preparation of difenoconazole antibody-time-resolved fluorescent microspheres: N-hydroxysuccinimide was added to 2-morpholine ethanesulfonic acid to prepare an N-hydroxysuccinimide solution with a concentration of 8-12 mg / mL for later use; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to 2-morpholine ethanesulfonic acid to prepare a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution with a concentration of 6-8 mg / mL for later use;
[0022] S32. Take 80-100 parts of N-hydroxysuccinimide solution and 80-100 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution and mix them thoroughly for later use.
[0023] S33. Take 10-15 parts of rare earth fluorescent complex microspheres and add them to the mixture in step S32. After mixing thoroughly, add 2.5-3.5 parts of difenoconazole antibody, mix thoroughly, and then couple the mixture. Activate the mixture at room temperature to obtain difenoconazole antibody-time-resolved fluorescent microspheres.
[0024] As a further embodiment of the present invention: in step S4, the concentration of bovine serum albumin is 0.05-0.06 mg / mL, the pH of the phosphate buffer is 7.2±1, the soaking time is 1-1.5 h, and the low-temperature drying temperature is 30-35 °C.
[0025] As a further embodiment of the present invention: the amount of difenoconazole antibody-time-resolved fluorescent microspheres sprayed in step S4 is 0.01 to 0.015 mL / cm.
[0026] As a further embodiment of the present invention: in step S5, the coating amount of the difenoconazole coating agent is 0.7-0.9 μL / cm, and the coating amount of the goat anti-mouse IgG antibody is 0.7-0.9 μL / cm.
[0027] As a further aspect of the present invention, the rare earth fluorescent complex microspheres contain one or more of Eu, Tb and Sm.
[0028] This invention provides an application of the time-resolved fluorescence immunochromatographic test strip described above in the detection of difenoconazole content in strawberries.
[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0030] This invention sequentially prepares a time-resolved fluorescent immunochromatographic test strip through the following steps: preparation of difenoconazole hapten, preparation of difenoconazole antibody, preparation of difenoconazole antibody-time-resolved fluorescent microspheres, preparation of fluorescent microsphere pads, preparation of reaction membrane, and assembly of test strip. This test strip exhibits high sensitivity for difenoconazole detection, capable of detecting low concentrations of difenoconazole in strawberries; it specifically detects difenoconazole in strawberries with high accuracy; and it also demonstrates good stability with a shelf life exceeding 12 months. Attached Figure Description
[0031] Figure 1 This is a schematic diagram of the structure of a time-resolved fluorescent immunochromatographic strip for detecting difenoconazole in strawberries. Detailed Implementation
[0032] Example 1:
[0033] In this embodiment of the invention, a method for preparing a time-resolved fluorescent immunochromatographic test strip for detecting difenoconazole in strawberries includes the following steps:
[0034] S1. Preparation of phenoxymethylconazole hapten: 4-(3-methyl-4-chlorophenoxyacetophenone) was dissolved in toluene in a round-bottom flask. 1,2-propanediol and p-toluenesulfonic acid were added, and the reaction was carried out at 120°C. The water produced in the reaction was separated using a water separator. The reaction was considered complete when no more water was produced. The reaction solution was cooled to room temperature. The lower layer of the reaction solution was separated using a separatory funnel, washed three times with saturated saline solution, and then twice with distilled water to obtain a pale yellow oily liquid, which is compound A. Compound A was dissolved in cyclohexane. One-third of a measured amount of bromine was added dropwise with stirring at room temperature. After the addition was complete, the red color of the reaction solution faded, and the temperature was lowered to 20°C. The addition continued until the addition was complete. The reaction was then heated to reflux in an oil bath and detected by TLC. After the reaction was complete, the reaction solution was cooled to room temperature, and the solvent was removed by vacuum distillation to obtain a yellowish-brown oily liquid, which was compound B. Compound B was dissolved in DMF, and potassium triazole salt was added. The reaction was carried out at 150°C and detected by TLC. The reaction was completed after 8 hours. DMF was recovered by vacuum distillation of the reaction solution. Water and toluene were then added, and the crude product was extracted using a separatory funnel. The product was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain a yellowish-brown solid. Recrystallization from ethanol yielded pale yellow needle-like crystals, which was compound C. Compound C was dissolved in acetonitrile, and an appropriate amount of water was added to make the solution just clear. Potassium permanganate was added in portions under ice bath conditions. After the addition was complete, the ice bath was removed, and the reaction was allowed to proceed at room temperature for 4 hours. The reaction solution was filtered, and the filter cake was washed twice with acetonitrile. The filtrate was collected, and the acetonitrile was removed by vacuum distillation. The residue was dissolved in ethyl acetate, purified by dry column chromatography, and the main UV-visible fraction was collected. After desolventization by vacuum distillation, a white solid was obtained, which was compound D. Compound D was dissolved in thionyl chloride, and 3 drops of DMF were added. The reaction was carried out at 6℃ for 4 hours. After the reaction was complete, the reaction solution was distilled under reduced pressure, and the remaining small amount of thionyl chloride was removed with n-hexane to obtain compound E. Compound E was dissolved in anhydrous tetrahydrofuran and cooled to 0℃ for later use. Separately, 4-aminobutyric acid was dissolved in 1N NaOH and cooled to 0℃. The tetrahydrofuran solution of compound E was added to the sodium hydroxide solution of 4-aminobutyric acid in 5 batches, with an interval of at least 10 minutes between each addition. After the addition was completed, the reaction was carried out in an ice bath. After about 3 hours, the reaction was complete. The reaction solution was distilled under reduced pressure to remove tetrahydrofuran. An appropriate amount of water was added to the residue, and the product was extracted three times with ethyl acetate. The organic phases were combined, dried with anhydrous sodium sulfate, and evaporated to dryness. The product was purified by silica gel column chromatography to obtain a pale yellow solid, which is the hapten of difenoconazole.
[0035] S2. Preparation of difenoconazole antibody: The difenoconazole hapten was conjugated with bovine serum albumin to obtain the difenoconazole immunogen; mice were then immunized with the difenoconazole immunogen, and then the mouse spleen cells and myeloma cells were fused and screened to obtain the difenoconazole antibody;
[0036] S3. Preparation of difenoconazole antibody-time-resolved fluorescent microspheres: N-hydroxysuccinimide was added to 2-morpholine ethanesulfonic acid to prepare an 8 mg / mL N-hydroxysuccinimide solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to 2-morpholine ethanesulfonic acid to prepare a 6 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution. 80 parts of the N-hydroxysuccinimide solution and 80 parts of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution were thoroughly mixed. 10 parts of rare earth fluorescent complex microspheres were added to the mixture from step S32. After thorough mixing, 2.5 parts of difenoconazole antibody were added and thoroughly mixed before coupling. The activation reaction was carried out at room temperature to obtain difenoconazole antibody-time-resolved fluorescent microspheres.
[0037] S4. Preparation of fluorescent microsphere pads: The polyester fiber layer was immersed in phosphate buffer containing 0.05 mg / mL bovine serum albumin for 1 h, and the pH was controlled at 7.2±1. After immersion, it was dried at 35℃. Then, the difenoconazole antibody-time-resolved fluorescent microspheres were uniformly sprayed onto the polyester fiber layer at a standard amount of 0.01 mL / cm. After drying at 35℃ again, the fluorescent microsphere pads were obtained.
[0038] S5. Preparation of the reaction membrane: The difenoconazole hapten is coupled with ovalbumin to obtain the difenoconazole coated antigen. The difenoconazole coated antigen is then coated onto a nitrocellulose membrane at a standard amount of 0.7 μL / cm to form the detection line, i.e., the T line. Goat anti-mouse IgG antibody is coated onto a nitrocellulose membrane at a standard amount of 0.7 μL / cm to form the quality control line, i.e., the C line, thus obtaining the reaction membrane.
[0039] S6. Test strip assembly: Attach the absorbent pad, reaction membrane, fluorescent microsphere pad, and sample pad to the base plate in sequence, then cut into 3mm wide test strips to obtain time-resolved fluorescence immunochromatographic test strips. Place them in a foil bag, add desiccant, and seal for storage. The attachment positions are shown in the attached diagram. Figure 1 As shown.
[0040] Example 2:
[0041] In this embodiment of the invention, a method for preparing a time-resolved fluorescent immunochromatographic test strip for detecting difenoconazole in strawberries includes the following steps:
[0042] S1. Preparation of phenoxymethylconazole hapten: 4-(3-methyl-4-chlorophenoxyacetophenone) was dissolved in toluene in a round-bottom flask. 1,2-propanediol and p-toluenesulfonic acid were added, and the reaction was carried out at 120°C. The water produced in the reaction was separated using a water separator. The reaction was considered complete when no more water was produced. The reaction solution was cooled to room temperature. The lower layer of the reaction solution was separated using a separatory funnel, washed three times with saturated saline solution, and then twice with distilled water to obtain a pale yellow oily liquid, which is compound A. Compound A was dissolved in cyclohexane. One-third of a measured amount of bromine was added dropwise with stirring at room temperature. After the addition was complete, the red color of the reaction solution faded, and the temperature was lowered to 20°C. The addition continued until the addition was complete. The reaction was then heated to reflux in an oil bath and detected by TLC. After the reaction was complete, the reaction solution was cooled to room temperature, and the solvent was removed by vacuum distillation to obtain a yellowish-brown oily liquid, which was compound B. Compound B was dissolved in DMF, and potassium triazole salt was added. The reaction was carried out at 150°C and detected by TLC. The reaction was completed after 8 hours. DMF was recovered by vacuum distillation of the reaction solution. Water and toluene were then added, and the crude product was extracted using a separatory funnel. The product was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain a yellowish-brown solid. Recrystallization from ethanol yielded pale yellow needle-like crystals, which was compound C. Compound C was dissolved in acetonitrile, and an appropriate amount of water was added to make the solution just clear. Potassium permanganate was added in portions under ice bath conditions. After the addition was complete, the ice bath was removed, and the reaction was allowed to proceed at room temperature for 4 hours. The reaction solution was filtered, and the filter cake was washed twice with acetonitrile. The filtrate was collected, and the acetonitrile was removed by vacuum distillation. The residue was dissolved in ethyl acetate, purified by dry column chromatography, and the main UV-visible fraction was collected. After desolventization by vacuum distillation, a white solid was obtained, which was compound D. Compound D was dissolved in thionyl chloride, and 3 drops of DMF were added. The reaction was carried out at 6℃ for 4 hours. After the reaction was complete, the reaction solution was distilled under reduced pressure, and the remaining small amount of thionyl chloride was removed with n-hexane to obtain compound E. Compound E was dissolved in anhydrous tetrahydrofuran and cooled to 0℃ for later use. Separately, 4-aminobutyric acid was dissolved in 1N NaOH and cooled to 0℃. The tetrahydrofuran solution of compound E was added to the sodium hydroxide solution of 4-aminobutyric acid in 5 batches, with an interval of at least 10 minutes between each addition. After the addition was completed, the reaction was carried out in an ice bath. After about 3 hours, the reaction was complete. The reaction solution was distilled under reduced pressure to remove tetrahydrofuran. An appropriate amount of water was added to the residue, and the product was extracted three times with ethyl acetate. The organic phases were combined, dried with anhydrous sodium sulfate, and evaporated to dryness. The product was purified by silica gel column chromatography to obtain a pale yellow solid, which is the hapten of difenoconazole.
[0043] S2. Preparation of difenoconazole antibody: The difenoconazole hapten was conjugated with bovine serum albumin to obtain the difenoconazole immunogen; mice were then immunized with the difenoconazole immunogen, and then the mouse spleen cells and myeloma cells were fused and screened to obtain the difenoconazole antibody;
[0044] S3. Preparation of difenoconazole antibody-time-resolved fluorescent microspheres: N-hydroxysuccinimide was added to 2-morpholine ethanesulfonic acid to prepare a 10 mg / mL N-hydroxysuccinimide solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to 2-morpholine ethanesulfonic acid to prepare a 7 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution. 90 parts of the N-hydroxysuccinimide solution and 90 parts of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution were thoroughly mixed. 12 parts of rare-earth fluorescent complex microspheres were added to the mixture from step S32. After thorough mixing, 3 parts of difenoconazole antibody were added and thoroughly mixed before coupling. The activation reaction was carried out at room temperature to obtain difenoconazole antibody-time-resolved fluorescent microspheres.
[0045] S4. Preparation of fluorescent microsphere pads: The polyester fiber layer was immersed in phosphate buffer containing 0.05 mg / mL bovine serum albumin for 1.2 h, and the pH was controlled at 7.2±1. After immersion, it was dried at 32℃. Then, the difenoconazole antibody-time-resolved fluorescent microspheres were uniformly sprayed onto the polyester fiber layer at a standard amount of 0.012 mL / cm. After drying at 32℃ again, the fluorescent microsphere pads were obtained.
[0046] S5. Preparation of the reaction membrane: The difenoconazole hapten is coupled with ovalbumin to obtain the difenoconazole coated antigen. The difenoconazole coated antigen is then coated onto a nitrocellulose membrane at a standard amount of 0.8 μL / cm to form the detection line, i.e., the T line. Goat anti-mouse IgG antibody is coated onto a nitrocellulose membrane at a standard amount of 0.8 μL / cm to form the quality control line, i.e., the C line, thus obtaining the reaction membrane.
[0047] S6. Test strip assembly: Attach the absorbent pad, reaction membrane, fluorescent microsphere pad, and sample pad to the base plate in sequence, then cut into 3mm wide test strips to obtain time-resolved fluorescence immunochromatographic test strips. Place them in a foil bag, add desiccant, and seal for storage. The attachment positions are shown in the attached diagram. Figure 1 As shown.
[0048] Example 3:
[0049] In this embodiment of the invention, a method for preparing a time-resolved fluorescent immunochromatographic test strip for detecting difenoconazole in strawberries includes the following steps:
[0050] S1. Preparation of phenoxymethylconazole hapten: 4-(3-methyl-4-chlorophenoxyacetophenone) was dissolved in toluene in a round-bottom flask. 1,2-propanediol and p-toluenesulfonic acid were added, and the reaction was carried out at 120°C. The water produced in the reaction was separated using a water separator. The reaction was considered complete when no more water was produced. The reaction solution was cooled to room temperature. The lower layer of the reaction solution was separated using a separatory funnel, washed three times with saturated saline solution, and then twice with distilled water to obtain a pale yellow oily liquid, which is compound A. Compound A was dissolved in cyclohexane. One-third of a measured amount of bromine was added dropwise with stirring at room temperature. After the addition was complete, the red color of the reaction solution faded, and the temperature was lowered to 20°C. The addition continued until the addition was complete. The reaction was then heated to reflux in an oil bath and detected by TLC. After the reaction was complete, the reaction solution was cooled to room temperature, and the solvent was removed by vacuum distillation to obtain a yellowish-brown oily liquid, which was compound B. Compound B was dissolved in DMF, and potassium triazole salt was added. The reaction was carried out at 150°C and detected by TLC. The reaction was completed after 8 hours. DMF was recovered by vacuum distillation of the reaction solution. Water and toluene were then added, and the crude product was extracted using a separatory funnel. The product was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain a yellowish-brown solid. Recrystallization from ethanol yielded pale yellow needle-like crystals, which was compound C. Compound C was dissolved in acetonitrile, and an appropriate amount of water was added to make the solution just clear. Potassium permanganate was added in portions under ice bath conditions. After the addition was complete, the ice bath was removed, and the reaction was allowed to proceed at room temperature for 4 hours. The reaction solution was filtered, and the filter cake was washed twice with acetonitrile. The filtrate was collected, and the acetonitrile was removed by vacuum distillation. The residue was dissolved in ethyl acetate, purified by dry column chromatography, and the main UV-visible fraction was collected. After desolventization by vacuum distillation, a white solid was obtained, which was compound D. Compound D was dissolved in thionyl chloride, and 3 drops of DMF were added. The reaction was carried out at 6℃ for 4 hours. After the reaction was complete, the reaction solution was distilled under reduced pressure, and the remaining small amount of thionyl chloride was removed with n-hexane to obtain compound E. Compound E was dissolved in anhydrous tetrahydrofuran and cooled to 0℃ for later use. Separately, 4-aminobutyric acid was dissolved in 1N NaOH and cooled to 0℃. The tetrahydrofuran solution of compound E was added to the sodium hydroxide solution of 4-aminobutyric acid in 5 batches, with an interval of at least 10 minutes between each addition. After the addition was completed, the reaction was carried out in an ice bath. After about 3 hours, the reaction was complete. The reaction solution was distilled under reduced pressure to remove tetrahydrofuran. An appropriate amount of water was added to the residue, and the product was extracted three times with ethyl acetate. The organic phases were combined, dried with anhydrous sodium sulfate, and evaporated to dryness. The product was purified by silica gel column chromatography to obtain a pale yellow solid, which is the hapten of difenoconazole.
[0051] S2. Preparation of difenoconazole antibody: The difenoconazole hapten was conjugated with bovine serum albumin to obtain the difenoconazole immunogen; mice were then immunized with the difenoconazole immunogen, and then the mouse spleen cells and myeloma cells were fused and screened to obtain the difenoconazole antibody;
[0052] S3. Preparation of difenoconazole antibody-time-resolved fluorescent microspheres: N-hydroxysuccinimide was added to 2-morpholine ethanesulfonic acid to prepare a 12 mg / mL N-hydroxysuccinimide solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to 2-morpholine ethanesulfonic acid to prepare an 8 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution. 100 parts of the N-hydroxysuccinimide solution and 100 parts of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution were thoroughly mixed. 15 parts of rare-earth fluorescent complex microspheres were added to the mixture from step S32. After thorough mixing, 3.5 parts of difenoconazole antibody were added and thoroughly mixed before coupling. The activation reaction was carried out at room temperature to obtain difenoconazole antibody-time-resolved fluorescent microspheres.
[0053] S4. Preparation of fluorescent microsphere pads: The polyester fiber layer was immersed in phosphate buffer containing 0.06 mg / mL bovine serum albumin for 1 h, and the pH was controlled at 7.2±1. After immersion, it was dried at 35℃. Then, the difenoconazole antibody-time-resolved fluorescent microspheres were uniformly sprayed onto the polyester fiber layer at a standard amount of 0.015 mL / cm. After drying at 35℃ again, the fluorescent microsphere pads were obtained.
[0054] S5. Preparation of the reaction membrane: The difenoconazole hapten is coupled with ovalbumin to obtain the difenoconazole coated antigen. The difenoconazole coated antigen is then coated onto a nitrocellulose membrane at a standard amount of 0.9 μL / cm to form the detection line, i.e., the T line. Goat anti-mouse IgG antibody is coated onto a nitrocellulose membrane at a standard amount of 0.9 μL / cm to form the quality control line, i.e., the C line, thus obtaining the reaction membrane.
[0055] S6. Test strip assembly: Attach the absorbent pad, reaction membrane, fluorescent microsphere pad, and sample pad to the base plate in sequence, then cut into 4mm wide test strips to obtain time-resolved fluorescence immunochromatographic test strips. Place them in a foil bag, add desiccant, and seal for storage. The attachment positions are shown in the attached diagram. Figure 1 As shown.
[0056] To better illustrate the technical effects of the present invention, the following experiments are conducted:
[0057] A hapten, complete antigen, antibody, and preparation method of difenoconazole disclosed in Chinese Patent No. CN114989144A; Publication Date: 2022-09-02 was selected as Comparative Example 1; a test strip and method for detecting difenoconazole disclosed in Chinese Patent No. CN112067814A; Publication Date: 2020-12-11 was selected as Comparative Example 2.
[0058] The test strips from Examples 1, 2, 3, Comparative Example 1, and Comparative Example 2 were used to detect difenoconazole in strawberries; the detection limit, specificity, false positive rate, false negative rate, and stability of the test strips were examined.
[0059] I. Detection Limit Test
[0060] Take blank strawberry samples and add difenoconazole at concentrations of 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.4 mg / kg, 0.6 mg / kg, 0.8 mg / kg, and 1.0 mg / kg, respectively. Then, use the test strips from Examples 1, 2, 3, Comparative Example 1, and Comparative Example 2 to detect the results. Observe the results and take the lowest standard concentration that only shows line C as the detection limit of the test strip.
[0061] The results showed that the detection limit of the test strips in Examples 1, 2, and 3 was 0.1 mg / kg; the detection limit of the test strip in Comparative Example 1 was 0.1 mg / kg; and the detection limit of the test strip in Comparative Example 2 was 0.4 mg / kg.
[0062] Therefore, it can be concluded that the test strip prepared by the present invention has a lower detection limit and higher detection sensitivity for difenoconazole, and can detect low concentrations of difenoconazole.
[0063] II. Specificity Test
[0064] Take blank strawberry samples and add metalaxyl, imidacloprid, triadimefon, triazole, hexaconazole, tebuconazole, and pendimethalin at concentrations of 0.05 mg / kg, 0.1 mg / kg, 0.4 mg / kg, 0.6 mg / kg, 0.8 mg / kg, and 1.0 mg / kg, respectively. Then, use the test strips from Examples 1, 2, 3, Comparative Example 1, and Comparative Example 2 to test the results. Observe the results and determine the specificity of the test strips based on the test results.
[0065] The results showed that the test strips in Examples 1, 2, 3, Comparative Example 1, and Comparative Example 2 did not cross-react with metalaxyl, imidacloprid, triadimefon, triazole, hexaconazole, tebuconazole, and tebuconazole, indicating good specificity. However, the test strip in Comparative Example 1 cross-reacted with triazole, hexaconazole, and tebuconazole, and the test strip in Comparative Example 2 cross-reacted with tebuconazole.
[0066] Therefore, it can be concluded that the test strip prepared in this invention can specifically detect difenoconazole in strawberries.
[0067] III. False Positive Rate and False Negative Rate Testing
[0068] Blank strawberry samples were taken. 100 samples were treated with 0.4 mg / kg difenoconazole and were designated as positive samples; the other 100 samples were treated without any drug and were designated as negative samples. The test strips from Examples 1, 2, 3, Comparative Example 1, and Comparative Example 2 were used for testing. The results were observed, the number of positive and negative samples was determined, and the false positive and false negative rates were calculated and recorded in Table 1 below.
[0069] Table 1. Analysis of False Positive and False Negative Rates
[0070]
[0071] From Table 1 above, it can be concluded that the false positive rate and false negative rate of the test strips in Examples 1, 2 and 3 are lower than those in Comparative Example 1 and Comparative Example 2; thus, it can be concluded that the test strips prepared by the present invention have high detection accuracy.
[0072] IV. Stability Testing
[0073] The test strips from Examples 1, 2, 3, Comparative Example 1, and Comparative Example 2 were placed at room temperature; the stability of difenoconazole in strawberries was tested monthly; the concentration of difenoconazole in positive strawberry samples was 0.4 mg / kg, and the positive strawberry samples were blank strawberries.
[0074] The results showed that in Examples 1, 2, and 3, the test results of negative strawberry samples were all negative and the test results of positive strawberry samples were all positive for the first 18 months, with a clear gradient. However, after 18 months, the stability of some test strips began to decline, and false positives occurred, while the stability of the test strips was good.
[0075] In Comparative Example 1, false positives appeared at 14 months; in Comparative Example 2, false positives appeared at 13 months.
[0076] Therefore, it can be concluded that the test strip prepared by this invention has good stability and can be stored for more than 12 months.
[0077] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A method for preparing a time-resolved fluorescent immunochromatographic test strip for detecting difenoconazole in strawberries, characterized in that, Includes the following steps: S1. Preparation of difenoconazole hapten: Using 4-(3-methyl-4-chlorophenyl)oxyacetophenone, 1,2-propanediol, p-toluenesulfonic acid, bromine, DMF, potassium triazole salt, potassium permanganate and sulfoxide, and after dissolution, distillation, crystallization and purification, difenoconazole hapten was obtained. S2. Preparation of difenoconazole antibody: The difenoconazole hapten was conjugated with bovine serum albumin to obtain the difenoconazole immunogen; mice were then immunized with the difenoconazole immunogen, and then the mouse spleen cells and myeloma cells were fused and screened to obtain the difenoconazole antibody; S3. Preparation of difenoconazole antibody-time-resolved fluorescent microspheres: N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added to 2-morpholine ethanesulfonic acid to prepare a mixed solution; then rare earth fluorescent complex microspheres and difenoconazole antibody were added to obtain difenoconazole antibody-time-resolved fluorescent microspheres. S4. Preparation of fluorescent microsphere pads: The polyester fiber layer was immersed in phosphate buffer containing bovine serum albumin. After immersion, it was dried at low temperature. Then, the difenoconazole antibody-time-resolved fluorescent microspheres were uniformly sprayed onto the polyester fiber layer and dried at low temperature again to obtain the fluorescent microsphere pads. S5. Preparation of reaction membrane: The difenoconazole hapten is coupled with ovalbumin to obtain the difenoconazole coated antigen, and then the difenoconazole coated antigen is coated onto a nitrocellulose membrane to form a detection line; the goat anti-mouse IgG antibody is coated onto a nitrocellulose membrane to form a quality control line, thus obtaining the reaction membrane; S6. Test strip assembly: Paste the absorbent pad, reaction membrane, fluorescent microsphere pad and sample pad onto the base plate in sequence, and then cut them into test strips 3-4 mm wide to obtain time-resolved fluorescence immunochromatographic test strips. Place them in a foil bag, add desiccant and seal for storage. The preparation method of the difenoconazole hapten in step S1 includes the following steps: S11. Dissolve 4-(3-methyl-4-chlorophenyl)oxyacetophenone in toluene in a round-bottom flask, add 1,2-propanediol and p-toluenesulfonic acid, raise the temperature to 120℃ and react. Use a water separator to separate the water produced in the reaction. When no more water is produced, the reaction is complete. Cool the reaction solution to room temperature, separate the lower layer of the reaction solution with a separatory funnel, wash it three times with saturated saline, and then wash it twice with distilled water to obtain a pale yellow oily liquid, which is compound A. S12. Dissolve compound A in cyclohexane, and add one-third of the measured amount of bromine dropwise while stirring at room temperature. After the addition is complete, wait for the red color of the reaction solution to disappear, and then lower the temperature to 20°C. Continue to add dropwise. After the addition is complete, heat the reaction solution in an oil bath under reflux and detect the reaction by TLC. After the reaction is complete, cool the reaction solution to room temperature and remove the solvent by vacuum distillation to obtain a yellowish-brown oily liquid, which is compound B. S13. Compound B was dissolved in DMF, potassium triazole salt was added, and the reaction was carried out at 150℃. The reaction was detected by TLC and the reaction was completed after 8 hours. The reaction solution was distilled under reduced pressure to recover DMF, and then water and toluene were added. The crude product was extracted with a separatory funnel, dried with anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure to obtain a yellowish-brown solid. The solid was recrystallized with ethanol to obtain pale yellow needle-like crystals, which is compound C. S14. Dissolve compound C in acetonitrile, add an appropriate amount of water to make the solution just clear, add potassium permanganate in portions under ice bath, remove the ice bath after the addition is complete, react at room temperature for 4 hours, filter the reaction solution, wash the filter cake twice with acetonitrile, take the filtrate, distill under reduced pressure to remove acetonitrile, dissolve the residue in ethyl acetate, dry sample loading and column purification, collect the main UV spot components, and after desolvation by reduced pressure distillation, obtain a white solid, which is compound D; S15. Compound D was dissolved in thionyl chloride, and 3 drops of DMF were added. The mixture was reacted at 116°C for 4 hours. After the reaction was complete, the reaction solution was distilled under reduced pressure, and the remaining small amount of thionyl chloride was removed with n-hexane to obtain compound E. Compound E was dissolved in anhydrous tetrahydrofuran and cooled to 0°C for later use. Separately, 4-aminobutyric acid was dissolved in 1N NaOH and cooled to 0°C. The tetrahydrofuran solution of compound E was added in 5 batches to the sodium hydroxide solution of 4-aminobutyric acid, with an interval of at least 10 minutes between each addition. After the addition was complete, the reaction was carried out in an ice bath for 3 hours. The reaction solution was then distilled under reduced pressure to remove the tetrahydrofuran. An appropriate amount of water was added to the residue, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness. The product was purified by silica gel column chromatography to obtain a pale yellow solid, which is the hapten of difenoconazole. The synthetic route of the difenoconazole hapten is as follows: 。 2. The method for preparing a time-resolved fluorescent immunochromatographic test strip for detecting difenoconazole in strawberries according to claim 1, characterized in that, The preparation method in step S3 includes the following steps: S31. Preparation of difenoconazole antibody-time-resolved fluorescent microspheres: N-hydroxysuccinimide was added to 2-morpholine ethanesulfonic acid to prepare an N-hydroxysuccinimide solution with a concentration of 8-12 mg / mL for later use; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to 2-morpholine ethanesulfonic acid to prepare a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution with a concentration of 6-8 mg / mL for later use; S32. Take 80-100 parts of N-hydroxysuccinimide solution and 80-100 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution and mix them thoroughly for later use. S33. Take 10-15 parts of rare earth fluorescent complex microspheres and add them to the mixture in step S32. After mixing thoroughly, add 2.5-3.5 parts of difenoconazole antibody, mix thoroughly, and then couple the mixture. Activate the mixture at room temperature to obtain difenoconazole antibody-time-resolved fluorescent microspheres.
3. The method for preparing a time-resolved fluorescent immunochromatographic test strip for detecting difenoconazole in strawberries according to claim 1, characterized in that, In step S4, the concentration of bovine serum albumin is 0.05–0.06 mg / mL, the pH of the phosphate buffer is 7.2 ± 1, and the soaking time is 1–1.5 h; the low-temperature drying temperature is 30–35 °C.
4. The method for preparing a time-resolved fluorescent immunochromatographic test strip for detecting difenoconazole in strawberries according to claim 1, characterized in that, In step S4, the amount of difenoconazole antibody-time-resolved fluorescent microspheres sprayed is 0.01–0.015 mL / cm.
5. The method for preparing a time-resolved fluorescent immunochromatographic test strip for detecting difenoconazole in strawberries according to claim 1, characterized in that, In step S5, the coating amount of the difenoconazole coating agent is 0.7–0.9 μL / cm, and the coating amount of the goat anti-mouse IgG antibody is 0.7–0.9 μL / cm.
6. The method for preparing a time-resolved fluorescent immunochromatographic test strip for detecting difenoconazole in strawberries according to claim 1, characterized in that, The rare earth fluorescent complex microspheres contain one or more of Eu, Tb, and Sm.
7. A method for preparing a time-resolved fluorescent immunochromatographic test strip for detecting difenoconazole in strawberries according to any one of claims 1-6, characterized in that, Application of the time-resolved fluorescence immunochromatographic test strip in the detection of difenoconazole content in strawberries.