A preparation method of a fluorescence immunochromatographic test strip for multiplex detection of silk and wool fabrics based on competitive method

Abstract

The present invention relates to the technical field of cultural relics detection, and discloses a preparation method of a fluorescence immunochromatographic test strip for multiplex detection of silk fabrics and wool fabrics based on a competitive method. In the present invention, polystyrene fluorescent microspheres are chemically combined with silk fibroin antibodies and wool keratin antibodies by the carbodiimide method. The first test line (T1 line) is sprayed with silk fibroin antigen, the second test line (T2 line) is sprayed with wool keratin antigen, and the quality control line (C line) is sprayed with goat anti-rabbit IgG to prepare a fluorescence immunochromatographic test strip.

Description

Technical Field

[0001] The present invention relates to the technical field of cultural relics detection, and particularly to a preparation method of a fluorescence immunochromatographic test strip for multiple detection of silk fabrics and wool fabrics based on a competitive method. Background Art

[0002] Since the new century, the scientific research investigation and excavation projects have increased exponentially. The unearthed textile cultural relics are of various types, covering a wide range, reflecting the ancient Chinese civilization and representing the scientific and cultural progress of human society. Among the unearthed textile cultural relics, protein-based textile cultural relics account for a large proportion. Studying the texture and species of textile cultural relics is of great significance for studying the development process of human civilization.

[0003] The underground environment where the protein-based cultural relic residues are located is relatively complex, with many impurities and low protein content. For these protein-based cultural relics with severely damaged morphology, it is difficult to identify them on-site using traditional detection methods, and a large amount of samples need to be taken. Therefore, finding a highly sensitive, specific, simple, fast, and efficient species identification method is of great significance for cultural relic protection and research.

[0004] Polystyrene fluorescent microspheres have attracted people's attention in enhancing the detection signal of portable test strips and improving sensitivity due to their structural stability, high light stability, and good safety. However, there has been no report on fluorescence immunochromatographic test strips for multiple detection of silk fabrics and wool fabrics. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a preparation method of a fluorescence immunochromatographic test strip for multiple detection of silk fabrics and wool fabrics based on a competitive method. The present invention chemically binds polystyrene fluorescent microspheres with silk fibroin antibody and wool keratin antibody through the carbodiimide method. The first test line (T1 line) is sprayed with silk fibroin antigen, the second test line (T2 line) is sprayed with wool keratin antigen, and the quality control line (C line) is sprayed with goat anti-rabbit IgG to prepare a fluorescence immunochromatographic test strip.

[0006] The specific technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a preparation method of a fluorescence immunochromatographic test strip for multiple detection of silk fabrics and wool fabrics based on a competitive method, including the following steps:

[0008] 1) Preparation of carboxyl-functionalized polystyrene fluorescent microspheres: Disperse polystyrene nanospheres in water containing SDS, then add CH2Cl2 containing a metal europium chelate fluorescent dye. Swell the polystyrene nanospheres by ultrasonic waves so that the metal europium chelate fluorescent dye is loaded into the swollen pores. After mixing, stir and rotary evaporate to remove CH2Cl2 to restore the swollen nanospheres to their original size, and then coat the dye. Then, centrifuge to collect the nanospheres loaded with the dye, wash with water to remove SDS, further wash and centrifuge with ethanol several times until no fluorescence is observed in the supernatant. Finally, dry the nanospheres and redisperse them in water. Take the obtained microsphere dispersion, add polyacrylic acid and perform ultrasonic treatment to carboxylate the surface of the microspheres. After centrifugal washing with water, carboxyl-functionalized polystyrene fluorescent microspheres are obtained for use.

[0009] 2) Preparation of polystyrene fluorescent microsphere-labeled silk fibroin antibody: Add carboxyl-functionalized polystyrene fluorescent microspheres to water for ultrasonic dispersion, then add N-hydroxysuccinimide ethanol solution and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution, shake well at room temperature, centrifuge, and discard the supernatant. After adding borate antibody coupling buffer for ultrasonic dispersion, slowly add silk fibroin antibody dropwise while stirring at room temperature. Stir at room temperature. After adding BSA for blocking, centrifuge, discard the supernatant, add preservation solution and mix well, and store in a refrigerator protected from light.

[0010] 3) Preparation of polystyrene fluorescent microsphere-labeled wool keratin antibody: Add carboxyl-functionalized polystyrene fluorescent microspheres to water for ultrasonic dispersion, then add N-hydroxysuccinimide ethanol solution and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution, shake well at room temperature, centrifuge, and discard the supernatant. After adding borate antibody coupling buffer for ultrasonic dispersion, slowly add wool keratin antibody dropwise while stirring at room temperature. Stir at room temperature. After adding BSA for blocking, centrifuge, discard the supernatant, add preservation solution and mix well, and store in a refrigerator protected from light.

[0011] 4) Assembly of the fluorescence immunochromatographic test strip: Use a membrane scribing machine to spray the silk fibroin antigen solution, wool keratin antigen solution, and goat anti-rabbit IgG solution prepared with PBS buffer solution onto the first test line (T1 line), the second test line (T2 line), and the quality control line (C line) of the nitrocellulose membrane respectively, and dry for standby. Coat the polystyrene fluorescent microsphere-labeled silk fibroin antibody and wool keratin antibody on the conjugate pad and dry for standby. Assemble the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad on the PVC bottom plate in sequence, and cut into strip-shaped test strips with a cutter, and store them dry and sealed.

[0012] Preferably, in step 1), the preparation of carboxyl-functionalized polystyrene fluorescent microspheres specifically includes: dispersing 90-110 mg of polystyrene nanospheres in 10 ml of water containing 0.02-0.03 mg of SDS, then adding 0.3-0.7 ml of CH2Cl2 containing 8-12 mg / mL of metal europium chelate fluorescent dye, swelling the polystyrene nanospheres by ultrasonic waves to load the metal europium chelate fluorescent dye into the swollen pores, after mixing for 50-70 s, stirring at 35-45 °C, rotary evaporating to remove CH2Cl2 to restore the swollen nanospheres to their original size, and then coating the dye; then centrifuging to collect the nanospheres loaded with the dye, washing with water to remove SDS, further washing and centrifuging with ethanol several times until no fluorescence is observed in the supernatant; finally drying the nanospheres and redispersing them in water at a concentration of 1.5-2.5 mg / mL; taking 1 mL of the obtained microsphere dispersion, adding 4-6 mg of polyacrylic acid and ultrasonically treating for 5-15 min to carboxylate the microsphere surface, and after centrifuging and washing with water, carboxyl-functionalized polystyrene fluorescent microspheres are obtained for use.

[0013] Preferably, in step 2), the preparation of polystyrene fluorescent microsphere-labeled silk fibroin antibody specifically includes: adding 0.3-0.7 mg of carboxyl-functionalized polystyrene fluorescent microspheres to 1 mL of water and ultrasonically dispersing for 15-25 min, then adding 4-6 μL each of 8-12 mg / mL of N-hydroxysuccinimide ethanol solution and 8-12 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution, shaking well at room temperature for 25-35 min, centrifuging at 13000-15000 rpm for 10-20 min, and removing the supernatant; adding borate antibody coupling buffer with pH = 7.8-8.2 and ultrasonically dispersing, then dropping 8-12 μg of silk fibroin antibody while stirring at room temperature for 0.5-1.5 h; adding BSA to a final concentration of 0.3-0.7% and blocking for 0.5-1.5 h, centrifuging at 12000-14000 rpm for 10-20 min, removing the supernatant, adding the preservation solution and mixing well, and storing refrigerated in the dark.

[0014] Preferably, in step 3), the preparation of the polystyrene fluorescent microsphere-labeled wool keratin antibody specifically includes: adding 0.3 - 0.7 mg of carboxyl-functionalized polystyrene fluorescent microspheres to 1 mL of water, ultrasonically dispersing for 15 - 25 min, then adding 4 - 6 μL each of 8 - 12 mg / mL N-hydroxysuccinimide ethanol solution and 8 - 12 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution, shaking well at room temperature for 25 - 35 min, centrifuging at 13000 - 15000 rpm for 10 - 20 min, and removing the supernatant; after adding borate antibody coupling buffer with pH = 8.8 - 9.2 and ultrasonically dispersing, while stirring, 13 - 17 μg of wool keratin antibody is added dropwise, and stirred at room temperature for 0.5 - 1.5 h; adding BSA to a final concentration of 0.3 - 0.7% and then blocking for 0.5 - 1.5 h, centrifuging at 12000 - 14000 rpm for 10 - 20 min, removing the supernatant, adding the preservation solution and mixing evenly, and storing refrigerated in the dark.

[0015] Preferably, in step 4), the assembly of the fluorescence immunochromatographic test strip specifically includes: using a membrane scribing machine to spray 0.8 - 1.2 mg / mL silk fibroin antigen solution, wool keratin antigen solution, and goat anti-rabbit IgG solution prepared with PBS buffer solution with pH = 7.4 on the first detection line (T1 line), the second detection line (T2 line), and the quality control line (C line) of a nitrocellulose membrane with a length of 2 - 3 cm and a width of 3 - 5 mm, drying at 35 - 39 °C for 10 - 15 h for standby; coating the silk fibroin antibody and wool keratin antibody labeled with polystyrene fluorescent microspheres on the conjugate pad, drying at 35 - 39 °C for 10 - 15 h for standby; sequentially assembling the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad on a PVC bottom plate, cutting into test strips with a width of 3 - 5 mm with a cutter, and storing in a dry and sealed manner.

[0016] In a second aspect, the present invention provides a method for detecting silk fabrics and wool fabrics using the above fluorescence immunochromatographic test strip: taking silk fabric and wool fabric trace samples, dissolving them in PBS buffer solution, stirring evenly, and then taking a drop of the supernatant and dropping it on the sample pad of the assembled fluorescence immunochromatographic test strip; if under ultraviolet light, the first detection line (T1 line) and the second detection line (T2 line) are colorless, and the quality control line (C line) shows red, it indicates that the sample contains silk fibroin and wool keratin, and the textile trace is a trace of silk fabric and wool fabric.

[0017] Preferably, the above detection method specifically includes: taking 1.5 - 2.5 mg of silk and wool fabric trace samples, dissolving them in 10 mL of PBS buffer solution with a pH of 7.4, stirring evenly, and after 1.5 - 2.5 h, taking a drop of the supernatant and dropping it on the sample pad of the assembled fluorescence immunochromatographic test strip; after 5 - 10 min, if the first detection line (T1 line) and the second detection line (T2 line) are colorless under ultraviolet light and the quality control line (C line) shows red, it indicates that the sample contains silk fibroin and wool keratin, and the textile trace is a trace of silk and wool fabrics.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] (1) When preparing carboxyl-functionalized polystyrene fluorescent microspheres in the present invention, the fluorescent dye enters the pores of the nanospheres through the swelling of the polystyrene nanospheres, and then the solvent is removed to restore the size of the nanospheres, thereby encapsulating the dye inside the nanospheres. This method is more stable compared to graft copolymerization of fluorescent materials with microspheres and is also more convenient in the preparation steps than synthesizing traditional core-shell structured fluorescent microspheres.

[0020] (2) The polystyrene microspheres containing metal chelates of europium element have characteristics such as large stokes shift, no inner quenching effect during aggregation, strong detection signal, and fluorescence stability, and are ideal markers for immunofluorescence chromatography.

[0021] (3) The present invention constructs a fluorescence immunochromatographic test strip for multiplex detection of silk and wool fabrics based on the competitive method. Polystyrene fluorescent microspheres are chemically combined with silk fibroin antibody and wool keratin antibody through the carbodiimide method. The first detection line (T1 line) is sprayed with silk fibroin antigen, the second detection line (T2 line) is sprayed with wool keratin antigen, and the quality control line (C line) is sprayed with goat anti-rabbit IgG to prepare an immunofluorescence chromatographic test strip with a competitive detection principle. Simultaneous detection of silk and wool fabrics is achieved. Description of the Drawings

[0022] Figure 1 It is a scanning electron microscope photograph of the polystyrene fluorescent microspheres prepared in Example 1;

[0023] Figure 2 It is a detection result diagram of the immunofluorescence chromatographic test strip prepared in Example 1 for textile and silk fabric trace samples. Detailed Embodiments

[0024] The present invention will be further described below in conjunction with embodiments.

[0025] Example 1

[0026] 1) Preparation of carboxyl-functionalized polystyrene fluorescent microspheres: A total of 100 mg of polystyrene nanospheres were dispersed in 10 ml of water (containing 0.025 mg of SDS), and then 0.5 ml of CH2Cl2 (10 mg / mL) containing a europium metal chelate fluorescent dye was added. The polystyrene nanospheres were swollen by ultrasonic waves to load the europium metal chelate fluorescent dye into the swollen pores of the nanospheres. After complete mixing for 1 minute, the solution was stirred at 40 °C, and CH2Cl2 was removed by rotary evaporation to restore the swollen microspheres to their original size, thereby encapsulating the dye in the pores into the nanospheres. Then, the prepared microspheres were collected by centrifugation, and the obtained microspheres were washed three times with water to remove SDS. Further washing and centrifugation with ethanol were performed several times until no fluorescence was observed in the supernatant. Finally, the microspheres were dried and redispersed in water at a concentration of 2 mg / mL. Take 1 mL of the dispersed microspheres, add 5 mg of polyacrylic acid, and perform carboxylation treatment on the surface of the microspheres by ultrasonic treatment for 10 min, and wash three times by centrifugation with water for later use.

[0027] 2) Preparation of polystyrene fluorescent microsphere-labeled fibroin antibody: 0.5 mg of carboxyl-functionalized polystyrene fluorescent microspheres were added to 1 mL of ultrapure water and ultrasonically dispersed for 20 min. Subsequently, 5 μL of freshly prepared N-hydroxysuccinimide ethanol solution (10 mg / mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution (10 mg / mL) were added, shaken well at room temperature for 30 min, centrifuged at 14000 rpm for 15 min, and the supernatant was removed. After ultrasonic dispersion in a borate antibody coupling buffer with pH = 8.0, 10 μg of fibroin antibody was added dropwise while stirring, and stirred at room temperature for 1 h. BSA (final concentration at 0.5%) was added for blocking for 1 h. Then, it was centrifuged at 13000 rpm for 15 min, the supernatant was removed, the preservation solution was added and mixed well, and stored at 4 °C in the dark for later use.

[0028] 3) Preparation of polystyrene fluorescent microsphere-labeled wool keratin antibody: 0.5 mg of polystyrene microspheres containing a europium metal chelate were added to 1 mL of ultrapure water and ultrasonically dispersed for 20 min. Subsequently, 5 μL of freshly prepared N-hydroxysuccinimide ethanol solution (10 mg / mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution (10 mg / mL) were added, shaken well at room temperature for 30 min, centrifuged at 14000 rpm for 15 min, and the supernatant was removed. After ultrasonic dispersion in a borate antibody coupling buffer with pH = 9.0, 15 μg of wool keratin antibody was added dropwise while stirring, and stirred at room temperature for 1 h. BSA (final concentration at 0.5%) was added for blocking for 1 h. Then, it was centrifuged at 13000 rpm for 15 min, the supernatant was removed, the preservation solution was added and mixed well, and stored at 4 °C in the dark for later use.

[0029] 4) Assembly of the test strip: Using a membrane - scribing machine, spray 1 mg / mL silk fibroin antigen, wool keratin antigen, and goat anti - rabbit IgG prepared with PBS buffer solution at pH = 7.4 onto the first test line (T1 line), the second test line (T2 line), and the quality control line (C line) of a nitrocellulose membrane that is 2.5 cm long and 4 mm wide, and dry it at 37 °C for 12 h for standby; Coat the silk fibroin antibody and wool keratin antibody labeled with polystyrene fluorescent microspheres on the conjugate pad, and dry it at 37 °C for 12 h for standby; Assemble the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad on the PVC bottom plate in sequence, cut it into test strips 4 mm wide with a cutter, and store it sealed in an aluminum foil bag with a desiccant.

[0030] 5) Take 2 mg of silk and wool textile trace samples, dissolve them in 10 mL of PBS buffer solution at pH 7.4, stir evenly, and after 2 h, take a drop of the supernatant and drop it on the sample pad of the assembled test strip; After 10 min, under ultraviolet light, the first test line (T1 line) and the second test line (T2 line) are colorless, and the quality control line (C line) shows red, indicating that the sample contains silk fibroin and wool keratin, and this textile trace is a trace of silk and wool textiles.

[0031] The scanning electron microscope photograph of the polystyrene fluorescent microspheres in Example 1 is as Figure 1 shown. The diameter of the polystyrene fluorescent microspheres is about 100 nm; The detection results of the immunofluorescence chromatography test strip for textile and silk textile trace samples are as Figure 2 shown. The negative control is PBS buffer solution at pH 7.4, and the detection result is as Figure 2 shown on the left. The detection results of textile and silk textile trace samples are as Figure 2 shown on the right. Under ultraviolet light, the first test line (T1 line) and the second test line (T2 line) are colorless, and the quality control line (C line) shows red, presenting a positive result.

[0032] Example 2

[0033] 1) Preparation of carboxyl-functionalized polystyrene fluorescent microspheres: Disperse a total of 100 mg of polystyrene nanospheres in 10 ml of water (containing 0.025 mg of SDS), then add 0.5 ml of CH2Cl2 (10 mg / mL) containing europium metal chelate fluorescent dye. Swell the polystyrene nanospheres by ultrasonic waves to load the europium metal chelate fluorescent dye into the swollen pores of the nanospheres. After complete mixing for 1 minute, stir the solution at 40 °C, and remove CH2Cl2 by rotary evaporation to restore the swollen microspheres to their original size, thereby encapsulating the dye in the pores into the nanospheres. This operation is more stable compared to graft copolymerization of fluorescent materials and microspheres, and is also more convenient than the steps for synthesizing traditional core-shell structure fluorescent microspheres; then centrifuge to collect the prepared microspheres, and wash the obtained microspheres three times with water to remove SDS; further wash and centrifuge several times with ethanol until no fluorescence is observed in the supernatant. Finally, dry the microspheres and redisperse them in water at a concentration of 2 mg / mL. Take 1 mL of the dispersed microspheres, add 5 mg of polyacrylic acid, and perform carboxylation treatment on the surface of the microspheres by ultrasonic treatment for 10 min, and wash three times by centrifugation with water for later use.

[0034] 2) Preparation of polystyrene fluorescent microsphere-labeled silk fibroin antibody: Add 1 mg of carboxyl-functionalized polystyrene fluorescent microspheres to 1 mL of ultrapure water and disperse them by ultrasonic treatment for 10 min. Subsequently, add 5 μL each of freshly prepared N-hydroxysuccinimide ethanol solution (10 mg / mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution (10 mg / mL), shake well at room temperature for 30 min, centrifuge at 12000 rpm for 15 min, and discard the supernatant; after adding borate antibody coupling buffer with pH = 8.0 and dispersing by ultrasonic treatment, slowly add 20 μg of silk fibroin antibody while stirring, and stir at room temperature for 1 h; add BSA (final concentration at 0.5%) for blocking for 1 h; then centrifuge at 13000 rpm for 15 min, discard the supernatant, add the preservation solution and mix well, and store at 4 °C in the dark for later use.

[0035] 3) Preparation of polystyrene fluorescent microsphere-labeled wool keratin antibody: Add 1 mg of polystyrene fluorescent microspheres containing europium metal chelate to 1 mL of ultrapure water and disperse them by ultrasonic treatment for 10 min. Subsequently, add 5 μL each of freshly prepared N-hydroxysuccinimide ethanol solution (10 mg / mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution (10 mg / mL), shake well at room temperature for 30 min, centrifuge at 12000 rpm for 15 min, and discard the supernatant; after adding borate antibody coupling buffer with pH = 9.0 and dispersing by ultrasonic treatment, slowly add 20 μg of wool keratin antibody while stirring, and stir at room temperature for 1 h; add BSA (final concentration at 0.5%) for blocking for 1 h; then centrifuge at 13000 rpm for 15 min, discard the supernatant, add the preservation solution and mix well, and store at 4 °C in the dark for later use.

[0036] 4) Assembly of the test strip: Use a membrane scribing machine to spray 1 mg / mL silk fibroin antigen, wool keratin antigen, and goat anti-rabbit IgG prepared with PBS buffer solution at pH = 7.4 onto the first test line (T1 line), the second test line (T2 line), and the quality control line (C line) of a nitrocellulose membrane that is 2.5 cm long and 4 mm wide, and dry it at 37 °C for 12 h for standby; coat the silk fibroin antibody and wool keratin antibody labeled with polystyrene fluorescent microspheres on the conjugate pad, and dry it at 37 °C for 8 h for standby; assemble the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad on the PVC bottom plate in sequence, cut it into test strips 4 mm wide with a cutter, and store it sealed in an aluminum foil bag with a desiccant.

[0037] 5) Take 2 mg of silk and wool fabric trace samples, dissolve them in 10 mL of PBS buffer solution at pH 7.4, stir evenly, and after 2 h, take a drop of the supernatant and drop it on the sample pad of the assembled test strip; after 10 min, under ultraviolet light, the first test line (T1 line) and the second test line (T2 line) are colorless, and the quality control line (C line) shows red, indicating that the sample contains silk fibroin and wool keratin, and this textile trace is the trace of silk and wool fabrics.

[0038] Example 3

[0039] 1) Preparation of carboxyl-functionalized polystyrene fluorescent microspheres: Disperse a total of 100 mg of polystyrene nanospheres in 10 ml of water (containing 0.025 mg of SDS), then add 0.5 ml of CH2Cl2 (10 mg / mL) containing a metal europium chelate fluorescent dye. Use ultrasonic waves to swell the polystyrene nanospheres so that the metal europium chelate fluorescent dye is loaded in the swollen pores of the nanospheres. After complete mixing for 1 minute, stir the solution at 40 °C, remove CH2Cl2 by rotary evaporation to restore the swollen microspheres to their original size, and then coat the dye in the pores into the nanospheres. This operation is more stable than graft copolymerization of fluorescent materials and microspheres, and is also more convenient than the steps for synthesizing traditional core-shell structured fluorescent microspheres; then centrifuge to collect the prepared microspheres, and wash the obtained microspheres three times with water to remove SDS; further wash and centrifuge with ethanol several times until no fluorescence is observed in the supernatant. Finally, dry the microspheres and redisperse them in water at a concentration of 2 mg / mL. Take 1 mL of the dispersed microspheres, add 5 mg of polyacrylic acid, and perform carboxylation treatment on the surface of the microspheres by ultrasonic treatment for 10 min, and wash them three times by centrifugation with water for standby.

[0040] 2) Preparation of polystyrene fluorescent microsphere-labeled silk fibroin antibody: Add 0.8 mg of carboxyl-functionalized polystyrene fluorescent microspheres into 1 mL of ultrapure water and disperse them by ultrasonic wave for 15 min. Then add 5 μL each of freshly prepared N-hydroxysuccinimide ethanol solution (10 mg / mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution (10 mg / mL), shake well at room temperature for 30 min, centrifuge at 14000 rpm for 15 min, and discard the supernatant. After adding borate antibody coupling buffer with pH = 8.0 and dispersing by ultrasonic wave, slowly add 15 μg of silk fibroin antibody dropwise while stirring, and stir at room temperature for 1 h. Add BSA (final concentration is 0.5%) for blocking for 1 h. Then centrifuge at 13000 rpm for 15 min, discard the supernatant, add preservation solution and mix well, and store in the dark at 4 °C for later use.

[0041] 3) Preparation of polystyrene fluorescent microsphere-labeled wool keratin antibody: Add 0.75 mg of polystyrene fluorescent microspheres containing europium element metal chelate into 1 mL of ultrapure water and disperse them by ultrasonic wave for 15 min. Then add 5 μL each of freshly prepared N-hydroxysuccinimide ethanol solution (10 mg / mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution (10 mg / mL), shake well at room temperature for 30 min, centrifuge at 14000 rpm for 15 min, and discard the supernatant. After adding borate antibody coupling buffer with pH = 9.0 and dispersing by ultrasonic wave, slowly add 20 μg of wool keratin antibody dropwise while stirring, and stir at room temperature for 1 h. Add BSA (final concentration is 0.5%) for blocking for 1 h. Then centrifuge at 13000 rpm for 15 min, discard the supernatant, add preservation solution and mix well, and store in the dark at 4 °C for later use.

[0042] 4) Assembly of test strip: Use a membrane scribing machine to spray 1 mg / mL silk fibroin antigen, wool keratin antigen, and goat anti-rabbit IgG prepared with PBS buffer solution with pH = 7.4 onto the first detection line (T1 line), the second detection line (T2 line), and the quality control line (C line) of a nitrocellulose membrane with a length of 2.5 cm and a width of 4 mm, and dry at 37 °C for 10 h for later use; Coat the polystyrene fluorescent microsphere-labeled silk fibroin antibody and wool keratin antibody on the conjugate pad, and dry at 37 °C for 10 h for later use; Assemble the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad on the PVC bottom plate in sequence, cut into test strips with a width of 4 mm with a cutter, and store them sealed in an aluminum foil bag with a desiccant.

[0043] 5) Take 2 mg of silk and wool fabric traces, dissolve them in 10 mL of PBS buffer solution with a pH of 7.4, stir evenly, and after 2 h, take a drop of the supernatant and drop it on the sample pad of the assembled test strip; after 10 min, under ultraviolet light, the first test line (T1 line) and the second test line (T2 line) are colorless, and the quality control line (C line) shows red, indicating that the sample contains silk fibroin and wool keratin, and the textile trace is a trace of silk and wool fabric.

[0044] The raw materials and equipment used in the present invention are all common raw materials and equipment in the art without special instructions; the methods used in the present invention are all conventional methods in the art without special instructions.

[0045] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent transformations made to the above embodiments according to the technical essence of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims

1. A preparation method of a fluorescence immunochromatographic test strip for multiple detection of silk and wool fabrics based on competitive law, characterized in that Including the following steps: 1) Preparation of carboxyl-functionalized polystyrene fluorescent microspheres: Disperse 90 - 110 mg of polystyrene nanospheres in 10 ml of water containing 0.02 - 0.03 mg of SDS, then add 0.3 - 0.7 ml of CH2Cl2 containing 8 - 12 mg / mL of metal europium chelate fluorescent dye. Swell the polystyrene nanospheres by ultrasonic waves to load the metal europium chelate fluorescent dye into the swollen pores. After mixing for 50 - 70 s, stir at 35 - 45 °C, and rotary evaporate to remove CH2Cl2 to restore the swollen nanospheres to their original size, thereby coating the dye. Then, centrifuge to collect the dye-loaded nanospheres, wash with water to remove SDS, further wash and centrifuge several times with ethanol until no fluorescence is observed in the supernatant. Finally, dry the nanospheres and redisperse them in water at a concentration of 1.5 - 2.5 mg / mL. Take 1 mL of the obtained microsphere dispersion, add 4 - 6 mg of polyacrylic acid and ultrasonically treat for 5 - 15 min to carboxylate the microsphere surface. After centrifugal washing with water, carboxyl-functionalized polystyrene fluorescent microspheres are obtained for use; 2) Preparation of polystyrene fluorescent microsphere-labeled silk fibroin antibody: Add the carboxyl-functionalized polystyrene fluorescent microspheres to water and ultrasonically disperse them. Subsequently, add N-hydroxysuccinimide ethanol solution and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution, shake well at room temperature, centrifuge, and discard the supernatant. After adding borate antibody coupling buffer and ultrasonically dispersing, slowly add silk fibroin antibody while stirring at room temperature. Stir at room temperature. After adding BSA for blocking, centrifuge, discard the supernatant, add preservation solution and mix well, store refrigerated in the dark; 3) Preparation of polystyrene fluorescent microsphere-labeled wool keratin antibody: Add the carboxyl-functionalized polystyrene fluorescent microspheres to water and ultrasonically disperse them. Subsequently, add N-hydroxysuccinimide ethanol solution and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution, shake well at room temperature, centrifuge, and discard the supernatant. After adding borate antibody coupling buffer and ultrasonically dispersing, slowly add wool keratin antibody while stirring at room temperature. Stir at room temperature. After adding BSA for blocking, centrifuge, discard the supernatant, add preservation solution and mix well, store refrigerated in the dark; 4) Assembly of the fluorescence immunochromatographic test strip: Use a membrane scribing machine to spray the silk fibroin antigen solution, wool keratin antigen solution, and goat anti-rabbit IgG solution prepared with PBS buffer solution on the first test line, second test line, and quality control line of the nitrocellulose membrane respectively, and dry for standby. Coat the polystyrene fluorescent microsphere-labeled silk fibroin antibody and wool keratin antibody on the conjugate pad and dry for standby. Assemble the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad on the PVC bottom plate in sequence, and cut into strip-shaped test strips with a cutter, and store them dry and sealed.

2. The preparation method according to claim 1, characterized in that: In step 2), the preparation of the polystyrene fluorescent microsphere-labeled silk fibroin antibody specifically includes: adding 0.3 - 0.7 mg of carboxyl-functionalized polystyrene fluorescent microspheres into 1 mL of water, ultrasonically dispersing for 15 - 25 min, then adding 4 - 6 μL each of 8 - 12 mg / mL N-hydroxysuccinimide ethanol solution and 8 - 12 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution, shaking well at room temperature for 25 - 35 min, centrifuging at 13000 - 15000 rpm for 10 - 20 min, and removing the supernatant; after adding borate antibody coupling buffer with pH = 7.8 - 8.2 and ultrasonically dispersing, adding 8 - 12 μg of silk fibroin antibody dropwise while stirring, and stirring at room temperature for 0.5 - 1.5 h; adding BSA to a final concentration of 0.3 - 0.7% and then blocking for 0.5 - 1.5 h, centrifuging at 12000 - 14000 rpm for 10 - 20 min, removing the supernatant, adding the preservation solution and mixing evenly, and storing refrigerated in the dark.

3. The preparation method according to claim 1, characterized in that: In step 3), the preparation of the polystyrene fluorescent microsphere-labeled wool keratin antibody specifically includes: adding 0.3 - 0.7 mg of carboxyl-functionalized polystyrene fluorescent microspheres into 1 mL of water, ultrasonically dispersing for 15 - 25 min, then adding 4 - 6 μL each of 8 - 12 mg / mL N-hydroxysuccinimide ethanol solution and 8 - 12 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution, shaking well at room temperature for 25 - 35 min, centrifuging at 13000 - 15000 rpm for 10 - 20 min, and removing the supernatant; after adding borate antibody coupling buffer with pH = 8.8 - 9.2 and ultrasonically dispersing, adding 13 - 17 μg of wool keratin antibody dropwise while stirring, and stirring at room temperature for 0.5 - 1.5 h; adding BSA to a final concentration of 0.3 - 0.7% and then blocking for 0.5 - 1.5 h, centrifuging at 12000 - 14000 rpm for 10 - 20 min, removing the supernatant, adding the preservation solution and mixing evenly, and storing refrigerated in the dark.

4. The preparation method according to claim 1, characterized in that: In step 4), the assembly of the fluorescence immunochromatographic test strip specifically includes: using a membrane scribing machine to spray 0.8 - 1.2 mg / mL silk fibroin antigen solution, wool keratin antigen solution, and goat anti-rabbit IgG solution prepared with PBS buffer solution with pH = 7.4 onto the first detection line, the second detection line, and the quality control line of a nitrocellulose membrane with a length of 2 - 3 cm and a width of 3 - 5 mm, drying at 35 - 39 °C for 10 - 15 h for standby; coating the polystyrene fluorescent microsphere-labeled silk fibroin antibody and wool keratin antibody on the conjugate pad, drying at 35 - 39 °C for 10 - 15 h for standby; sequentially assembling the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent pad on a PVC bottom plate, cutting into test strips with a width of 3 - 5 mm with a cutter, and storing in a dry and sealed manner.

5. A method for detecting silk fabrics and wool fabrics using a fluorescence immunoassay chromatography test strip obtained by the preparation method described in any one of claims 1-4, characterized in that: Take silk and wool fabric trace samples, dissolve them in PBS buffer solution, stir evenly, and then take a drop of the supernatant and drop it on the sample pad of the assembled fluorescence immunochromatographic test strip; if the first test line and the second test line are colorless under ultraviolet light and the control line shows red, it indicates that the sample contains fibroin and wool keratin, and the trace is the trace of silk and wool fabrics.

6. The method according to claim 5, characterized in that: Specifically include: Take 1.5 - 2.5 mg of silk and wool fabric trace samples, dissolve them in 10 mL of PBS buffer solution with a pH of 7.4, stir evenly, and after 1.5 - 2.5 h, take a drop of the supernatant and drop it on the sample pad of the assembled fluorescence immunochromatographic test strip; after 5 - 10 min, if the first test line and the second test line are colorless under ultraviolet light and the control line shows red, it indicates that the sample contains fibroin and wool keratin, and the trace is the trace of silk and wool fabrics.

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