A preparation method of a fluorescence test strip for detecting fibroin based on the double antibody sandwich method

Abstract

The present invention relates to the technical field of cultural relics detection, and discloses a preparation method of a fluorescence test strip for detecting fibroin based on a double-antibody sandwich method. The present invention constructs a fluorescence test strip for detecting fibroin based on a double-antibody sandwich method. Through the chemical combination of the carboxyl groups on the surface of polystyrene fluorescent microspheres and the amino groups on the surface of fibroin monoclonal antibodies, a polyclonal antibody of fibroin is sprayed on the test line, and goat anti-mouse IgG is sprayed on the quality control line to prepare a fluorescence immunochromatographic test strip based on the double-antibody sandwich detection principle. When detecting, different from the color development of the traditional colloidal gold competition method, if two lines show color simultaneously, it is positive, and if only the quality control line shows color, it is negative.

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 test strip for detecting fibroin based on the double antibody sandwich method. Background Art

[0002] China has always had the beautiful name of the "Silk Country". Silk has not only been used as a silk fabric for clothing, but also served as a carrier of Eastern civilization to strengthen cultural exchanges between the East and the West. Since the new century, the number of scientific research investigation and excavation projects has increased exponentially, and various natural science technologies such as archaeological dating, material structure and chemical composition analysis have been increasingly widely applied to Chinese archaeological research.

[0003] More and more silk fabrics have been unearthed from tombs. Among the currently unearthed textiles, protein-based textiles account for the majority. Such textiles are easily affected by moisture, temperature, acidity and alkalinity, and microorganisms in the tomb environment and undergo aging and decomposition, resulting in the breakage of macromolecular chains and the degradation of peptide segments, and have become fragments and microtraces when unearthed. By constructing new technical methods to identify these microtraces of silk fabrics, the emotions and cultural values of people at that time can be restored, and at the same time, important technical support can be provided for exploring the origin and development of textiles.

[0004] The traditional colloidal gold competition method for test strips has poor stability, and the color development method is also different from the conventional idea. The disappearance of the test line can prove that the sample contains the target analyte. Therefore, there is a need for a portable test strip with stable marker testing and more significant and clear test results. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a preparation method of a fluorescence test strip for detecting fibroin based on the double antibody sandwich method. The present invention chemically combines polystyrene fluorescent microspheres with fibroin monoclonal antibodies, sprays fibroin polyclonal antibodies on the test line, and sprays goat anti-mouse IgG on the quality control line to prepare a fluorescence immunochromatographic test strip. When detected by the present invention, it is different from the color development of the traditional colloidal gold competition method. When both lines show color simultaneously, it is positive, and when only the quality control line shows color, it is negative.

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

[0007] In the first aspect, the present invention provides a preparation method of a fluorescence test strip for detecting fibroin based on the double antibody sandwich method, including the following steps:

[0008] 1) Preparation of carboxyl-functionalized polystyrene fluorescent microspheres: Dispersed polystyrene nanospheres in water containing SDS, then added CH2Cl2 containing metal europium chelate fluorescent dye. The polystyrene nanospheres were swollen by ultrasonic waves to load the metal europium chelate fluorescent dye into the swollen pores. After mixing, stirred, and removed CH2Cl2 by rotary evaporation to restore the swollen nanospheres to their original size, and then the dye was coated. Then, the nanospheres loaded with the dye were collected by centrifugation, washed with water to remove SDS, further washed and centrifuged with ethanol several times until no fluorescence was observed in the supernatant. Finally, the nanospheres were dried and redispersed in water. Take the obtained microsphere dispersion, add polyacrylic acid and perform ultrasonic treatment to carboxylate the microsphere surface. After centrifugal washing with water, carboxyl-functionalized polystyrene fluorescent microspheres were obtained for use.

[0009] 2) Preparation of silk fibroin monoclonal antibody labeled with polystyrene fluorescent microspheres: 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 remove the supernatant. After ultrasonic dispersion with borate antibody coupling buffer, add silk fibroin monoclonal antibody dropwise with stirring at room temperature and stir at room temperature. Add BSA silicone oil for blocking; centrifuge, remove the supernatant, add preservation solution and mix well, and store refrigerated in the dark.

[0010] 3) Assembly of test strips: Use a membrane scribing machine to spray silk fibroin polyclonal antibody solution and goat anti-mouse IgG solution prepared with PBS buffer solution on the test line and quality control line of the nitrocellulose membrane respectively, and dry for standby; Coat the silk fibroin monoclonal antibody labeled with polystyrene fluorescent microspheres diluted with borate buffer solution 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, cut into wide test strips with a cutter, and store them dry and sealed.

[0011] Preferably, in step 1), the dosage ratio of polystyrene nanospheres, water containing SDS, and CH2Cl2 containing 8-12 mg / mL of metal europium chelate fluorescent dye is 90-110 mg: 10 ml: 0.3-0.7 ml.

[0012] Preferably, in step 1), the concentration of SDS in the water containing SDS is 0.02-0.03 mg; the concentration of the metal europium chelate fluorescent dye in the CH2Cl2 containing the metal europium chelate fluorescent dye is 8-12 mg / mL.

[0013] Preferably, in step 1), the mixing time is 50-70 s, and the stirring temperature is 35-45 °C.

[0014] Preferably, in step 1), the nanospheres are finally dried and redispersed in water at a concentration of 1.5 - 2.5 mg / mL; 1 mL of the obtained microsphere dispersion is taken, 4 - 6 mg of polyacrylic acid is added, and ultrasonic treatment is carried out for 5 - 15 min to carboxylate the surface of the microspheres. After centrifugal washing with water, carboxyl-functionalized polystyrene fluorescent microspheres are obtained for use.

[0015] Preferably, in step 2), the dosage ratio of the carboxyl-functionalized polystyrene fluorescent microspheres, water, N-hydroxysuccinimide ethanol solution, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution is 0.3 - 0.7 mg: 1 mL: 3 - 7 μL: 3 - 7 μL.

[0016] Preferably, in step 2), the concentrations of the N-hydroxysuccinimide ethanol solution and the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution are respectively 8 - 12 mg / mL.

[0017] Preferably, in step 2), the time for shaking at room temperature is 20 - 40 min, and the first centrifugation conditions are 13000 - 15000 rpm for 10 - 20 min.

[0018] Preferably, in step 2), the pH of the borate antibody conjugation buffer is 7.8 - 8.2; the addition amount of the silk fibroin monoclonal antibody is 8 - 12 μg.

[0019] Preferably, in step 2), the stirring time at room temperature is 0.5 - 1.5 h; the final concentration of BSA is 0.3 - 0.7 wt%, and the blocking time is 0.5 - 1.5 h; the second centrifugation conditions are 12000 - 14000 rpm for 10 - 20 min.

[0020] Preferably, in step 3), the pH of the PBS buffer solution is 7.3 - 7.5, and the concentrations of the silk fibroin polyclonal antibody solution and the goat anti-mouse IgG solution are 0.8 - 1.2 mg / mL.

[0021] Preferably, in step 3), the length of the nitrocellulose membrane is 2.3 - 2.7 cm and the width is 3 - 5 mm; the drying conditions at 37 °C are 35 - 39 °C for 10 - 15 h; for standby.

[0022] Preferably, in step 3), the pH of the borate buffer solution is 7.8 - 8.2.

[0023] Preferably, in step 3), the width of the test strip is 3 - 5 mm.

[0024] Second aspect, the present invention provides a method for detecting textiles using the above-mentioned fluorescent test strip: Take a textile trace sample, dissolve it in PBS buffer solution, stir evenly, and take a drop of the supernatant and drop it on the sample pad of the assembled test strip; If both the test line and the quality control line show red under ultraviolet light, it indicates that the sample contains silk fibroin, and this textile trace is a trace of silk fabric.

[0025] The specific test method is: Take 1.5 - 2.5 mg of the textile trace sample, dissolve it in 8 - 12 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 test strip; After 5 - 10 min, if both the test line and the quality control line show red under ultraviolet light, it indicates that the sample contains silk fibroin, and this textile trace is a trace of silk fabric.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] (1) When preparing the 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 than graft copolymerization of fluorescent materials and microspheres, and is also more convenient in the preparation steps than synthesizing traditional core-shell structured fluorescent microspheres.

[0028] (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.

[0029] (3) The present invention constructs a fluorescent test strip for detecting silk fibroin based on the double antibody sandwich method. Through the chemical combination of the carboxyl groups on the surface of the polystyrene fluorescent microspheres and the amino groups on the surface of the silk fibroin monoclonal antibody, the silk fibroin polyclonal antibody is sprayed on the test line, and the goat anti-mouse IgG is sprayed on the quality control line to prepare a fluorescent immunochromatographic test strip based on the double antibody sandwich detection principle. Different from the color development of the traditional colloidal gold competition method during the detection of the present invention, when both lines show color simultaneously, it is positive, and when only the quality control line shows color, it is negative.

[0030] (4) The present invention requires less sample amount and can detect silk fibroin quickly, intuitively, accurately, and with high sensitivity, which is of great significance for detecting severely degraded silk fabric cultural relics. Description of the Drawings

[0031] Figure 1 Transmission electron microscope photograph of the polystyrene fluorescent microspheres labeled with silk fibroin monoclonal antibody prepared in Example 1;

[0032] Figure 2Detection result diagram of the fluorescent test strip prepared in Example 1 for textile trace samples. Detailed implementation mode

[0033] The present invention will be further described below in conjunction with embodiments.

[0034] Example 1

[0035] 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 standby.

[0036] 2) Preparation of polystyrene fluorescent microsphere-labeled silk fibroin monoclonal antibody: 0.5 mg of polystyrene fluorescent 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, and the mixture was shaken at room temperature for 30 min and centrifuged at 14000 rpm for 15 min to remove the supernatant. After ultrasonic dispersion in a borate antibody coupling buffer with pH = 8.0, 10 μg of silk fibroin monoclonal antibody was added dropwise while stirring, and the mixture was stirred at room temperature for 1 h. BSA (final concentration 0.5%) was added for blocking for 1 h. Then, it was centrifuged at 13000 rpm for 15 min to remove the supernatant, and the preservation solution was added and mixed evenly, and stored at 4 °C in the dark for standby.

[0037] 3) Assembly of the test strip: Use a membrane scribing machine to spray a 1 mg / mL silk fibroin polyclonal antibody solution and a goat anti-mouse IgG solution prepared with PBS buffer solution at pH = 7.4 onto the test line and the quality control 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 monoclonal antibody labeled with polystyrene fluorescent microspheres diluted with borate buffer solution at pH 8.0 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.

[0038] 4) Take 2 mg of textile trace sample, dissolve it 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, both the test line and the quality control line show red under ultraviolet light, indicating that the sample contains silk fibroin, and this textile trace is a trace of silk fabric.

[0039] The transmission electron microscope photograph of the silk fibroin monoclonal antibody labeled with polystyrene fluorescent microspheres prepared in Example 1 is as Figure 1 shown. The diameter of the polystyrene fluorescent microspheres is about 100 nm, and macromolecular antibody proteins are evenly bound around them; The detection result of the fluorescent immunochromatographic test strip for the textile trace sample is as Figure 2 shown. The detection result of the textile trace sample is as Figure 2 shown on the left. Both the test line and the quality control line show red under ultraviolet light, presenting a positive result. The negative control is PBS buffer solution at pH 7.4, and the detection result is as Figure 2 shown on the right.

[0040] Example 2

[0041] 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 metal europium chelate fluorescent dye was added. The polystyrene nanospheres were swollen by ultrasonic waves to allow the metal europium chelate fluorescent dye to be loaded 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. 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 the prepared microspheres were collected by centrifugation, and the obtained microspheres were washed three times with water to remove SDS; further washed and centrifuged several times with ethanol 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 with water by centrifugation for later use.

[0042] 2) Preparation of polystyrene fluorescent microsphere-labeled silk fibroin monoclonal antibody: 0.75 mg of polystyrene fluorescent microspheres containing a metal chelate of europium element were added to 1 mL of ultrapure water and ultrasonically dispersed for 15 min. Subsequently, 2.5 μL each of freshly prepared N-hydroxysuccinimide ethanol solution (10 mg / mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution (10 mg / mL) were added, shaken well at room temperature for 30 min, centrifuged at 12000 rpm for 15 min, and the supernatant was removed; after ultrasonic dispersion in a borate antibody coupling buffer with pH = 8.0, 18 μg of silk fibroin monoclonal antibody was added dropwise with stirring at room temperature, and stirred for 1 h at room temperature; BSA (final concentration at 0.5%) was added for blocking for 1 h; then centrifuged at 13000 rpm for 15 min, the supernatant was removed, the preservation solution was added and mixed well, and stored refrigerated in the dark at 4 °C for later use.

[0043] 3) Assembly of test strips: A 1 mg / mL silk fibroin polyclonal antibody solution and a goat anti-mouse IgG solution prepared with a PBS buffer solution with pH = 7.4 were respectively sprayed on the test line and the quality control line of a nitrocellulose membrane with a length of 2.5 cm and a width of 4 mm using a membrane scribing machine, and dried at 37 °C for 10 h for later use; the polystyrene fluorescent microsphere-labeled silk fibroin monoclonal antibody diluted with a borate buffer solution with pH = 8.0 was coated on the conjugate pad and dried at 37 °C for 10 h for later use; the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad were sequentially assembled on a PVC bottom plate, cut into test strips with a width of 4 mm with a cutter, and stored sealed in an aluminum foil bag with a desiccant.

[0044] 4) Take 2 mg of the textile trace sample, dissolve it in 10 mL of PBS buffer solution with a pH of 7.4, stir well, 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, both the test line and the control line show red under ultraviolet light, indicating that the sample contains fibroin protein and the textile trace is a trace of silk fabric.

[0045] Example 3

[0046] 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. Ultrasonic waves are used to swell the polystyrene nanospheres so that the metal europium chelate fluorescent dye is loaded in the pores of the swollen nanospheres. After complete mixing for 1 minute, the solution is stirred at 40 °C, and CH2Cl2 is removed 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 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 the prepared microspheres are collected by centrifugation, and the obtained microspheres are washed three times with water to remove SDS; further washed and centrifuged several times with ethanol until no fluorescence is observed in the supernatant. Finally, the microspheres are 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 ultrasonically treat for 10 min to carboxylate the surface of the microspheres, and wash three times by centrifugation with water for later use.

[0047] 2) Preparation of polystyrene fluorescent microsphere-labeled fibroin monoclonal antibody: Add 1 mg of polystyrene fluorescent microspheres containing a metal chelate of europium element to 1 mL of ultrapure water and ultrasonically disperse 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 remove the supernatant; after adding borate antibody coupling buffer with pH = 8.0 and ultrasonically dispersing, 20 μg of fibroin monoclonal antibody is added dropwise 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, remove the supernatant, add the preservation solution and mix well, and store in a refrigerator at 4 °C in the dark for later use.

[0048] 3) Assembly of the test strip: Use a membrane scribing machine to spray the 1 mg / mL silk fibroin polyclonal antibody solution and goat anti-mouse IgG solution prepared with PBS buffer solution at pH = 7.4 onto the test line and quality control 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 monoclonal antibody labeled with polystyrene fluorescent microspheres diluted with borate buffer solution at pH 8.0 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 a test strip 4 mm wide with a cutter, and store it sealed in an aluminum foil bag with a desiccant.

[0049] 4) Take 5 mg of textile trace sample, dissolve it 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, both the test line and the quality control line show red under ultraviolet light, indicating that the sample contains silk fibroin, and this textile trace is a trace of silk fabric.

[0050] 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.

[0051] 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 fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a fluorescence test strip for detecting silk fibroin based on the double antibody sandwich method, characterized in that It includes the following steps: 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 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 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. 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. The dosage ratio of polystyrene nanospheres, water containing SDS, and CH2Cl2 containing 8 - 12 mg / mL of metal europium chelate fluorescent dye is 90 - 110 mg: 10 ml: 0.3 - 0.7 ml. The concentration of SDS in the water containing SDS is 0.02 - 0.03 mg; the concentration of the metal europium chelate fluorescent dye in the CH2Cl2 containing the metal europium chelate fluorescent dye is 8 - 12 mg / mL. 2) Preparation of polystyrene fluorescent microsphere-labeled silk fibroin monoclonal antibody: Ultrasonically disperse the carboxyl-functionalized polystyrene fluorescent microspheres in water, 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 ultrasonic dispersion with borate antibody coupling buffer, slowly add the silk fibroin monoclonal antibody dropwise while stirring at room temperature. Stir at room temperature. Add BSA silicone oil for blocking. Centrifuge, discard the supernatant, add the preservation solution and mix well, and store in the dark at low temperature. 3) Assembly of the test strip: Use a membrane scribing machine to spray the silk fibroin polyclonal antibody solution and goat anti-mouse IgG solution prepared with PBS buffer solution on the test line and the quality control line of the nitrocellulose membrane respectively, and dry for later use. Coat the silk fibroin monoclonal antibody labeled with polystyrene fluorescent microspheres diluted with borate buffer solution on the conjugate pad and dry for later use. Assemble the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad on the PVC bottom plate in sequence, and cut into wide 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 dosage ratio of carboxyl-functionalized polystyrene fluorescent microspheres, water, N-hydroxysuccinimide ethanol solution, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution is 0.3 - 0.7 mg: 1 mL: 3 - 7 μL: 3 - 7 μL. The concentrations of the N-hydroxysuccinimide ethanol solution and the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ethanol solution are 8 - 12 mg / mL respectively.

3. The preparation method according to claim 2, characterized in that: In step 2), the time for shaking well at room temperature is 20 - 40 min, and the conditions for the first centrifugation are 13000 - 15000 rpm for 10 - 20 min.

4. The preparation method according to claim 3, characterized in that: In step 2), The pH of the borate antibody coupling buffer is 7.8 - 8.2; the addition amount of the silk fibroin monoclonal antibody is 8 - 12 μg; The stirring time at room temperature is 0.5 - 1.5 h; the final concentration of BSA is 0.3 - 0.7 wt%, and the blocking time is 0.5 - 1.5 h; the conditions for the second centrifugation are 12000 - 14000 rpm for 10 - 20 min.

5. The preparation method according to claim 4, characterized in that: In step 3), The pH of the PBS buffer solution is 7.3 - 7.5, and the concentrations of the silk fibroin polyclonal antibody solution and the goat anti - mouse IgG solution are 0.8 - 1.2 mg / mL; The length of the nitrocellulose membrane is 2.3 - 2.7 cm and the width is 3 - 5 mm; the drying conditions at 37℃ are 35 - 39℃ for 10 - 15 h; reserve for use; The pH of the borate buffer solution is 7.8 - 8.2; The width of the test strip is 3 - 5 mm.

6. A method for detecting textiles using a fluorescent test strip obtained by the preparation method according to any one of claims 1-5, characterized in that: Take the textile trace sample, dissolve it in the PBS buffer solution, stir evenly, and take a drop of the supernatant and drop it on the sample pad of the assembled test strip; if both the test line and the quality control line show red under ultraviolet light, it indicates that the sample contains silk fibroin, and this textile trace is a trace of silk fabric.

7. The method according to claim 6, wherein: Take 1.5 - 2.5 mg of the textile trace sample, dissolve it in 8 - 12 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 test strip; after 5 - 10 min, if both the test line and the quality control line show red under ultraviolet light, it indicates that the sample contains silk fibroin, and this textile trace is a trace of silk fabric.

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