Preparation method and application of an immunomagnetic bead test strip for rapid detection of trace proteins in cultural relics

Abstract

The present invention relates to the technical field of the detection of ancient silk fabrics, and discloses a preparation method and application of an immunomagnetic bead test strip for rapidly detecting trace proteins in cultural relics, comprising the following steps: (1) after the carboxyl magnetic beads are subjected to a coupling reaction with protein antibodies, the supernatant is removed by magnetic separation, and after washing with a buffer solution and a cleaning agent in sequence, immunomagnetic beads are obtained; (2) the immunomagnetic beads are sprayed on a pretreated nitrocellulose membrane, and after drying, they are reserved for use; the diluted antibody solution I and antibody solution II are respectively sprayed on the test line and the quality control line of the nitrocellulose membrane, and after drying, they are reserved for use; (3) a sample pad, an immunomagnetic bead conjugate pad, a nitrocellulose membrane and a water absorption pad are assembled and cut in sequence to obtain an immunomagnetic bead test strip, which is stored in a dry and sealed manner. The present invention can achieve rapid and sensitive detection while enriching trace proteins, is convenient for operation at the archaeological site, and takes a short time.

Description

Technical Field

[0001] The present invention relates to the technical field of protein detection in ancient cultural relics, and in particular to a preparation method and application of an immunomagnetic bead test strip for rapidly detecting trace proteins in cultural relics. Background Art

[0002] Ancient cultural relics include silk cultural relics and leather cultural relics, the main components of which are all proteins. Buried in the underground tomb environment for a long time, affected by factors such as humidity, heat and microorganisms, the fibers will degrade to varying degrees. After the cultural relics are unearthed, due to the drastic change of environmental conditions, the aging of the cultural relics is further accelerated. Therefore, in the long historical process, some cultural relics buried in tombs or sites in those days have already lost their physical appearance, degraded into peptide segments and amino acids, or become traces, or melted into the soil, and can no longer be identified by the naked eye. With more and more early sites being excavated, only by using modern advanced natural science means to extract information of ancient cultural relics from the fragments can more archaeological evidences be provided for the research on the origin and dissemination process of silk and leather cultural relics. Therefore, the development of rapid detection technology at the archaeological site is very crucial. However, the protein content in imprints, residues and soil is extremely low. Therefore, before detecting cultural relics, it is necessary to enrich the trace proteins in them to improve the detection sensitivity, and at the same time, issues such as how to simplify the operation and shorten the time-consuming need to be considered.

[0003] The Chinese invention patent with the publication number CN108387435B discloses a method for enriching trace fibroin proteins. This method can enrich trace fibroin proteins through immunomagnetic beads, but after enrichment, it needs to be eluted and then detected by ELISA. The process is relatively complex and time-consuming, and it is not suitable for rapid detection at the archaeological site. The Chinese invention patent with the publication number CN104459162B discloses a preparation method of an indirect competition method detection test paper for ancient silk fabrics, and the Chinese invention patent with the publication number CN104459118B discloses a preparation method of a double antibody sandwich method detection test paper for ancient silk fabrics. Although the test paper can rapidly detect the residues of silk fabrics at the archaeological site, a detection method with higher sensitivity is required for detecting residues with low content at the archaeological site. Summary of the Invention

[0004] In order to solve the technical problem of how to rapidly detect trace proteins at the archaeological site, the present invention provides a preparation method and application of an immunomagnetic bead test strip for rapidly detecting trace proteins in cultural relics. By combining magnetic beads with immunochromatography technology, it can rapidly detect trace proteins at the archaeological site, and the operation is convenient and the time-consuming is short.

[0005] The specific technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for preparing an immunomagnetic bead test strip for rapidly detecting trace proteins in cultural relics, comprising the following steps:

[0007] (1) After the carboxyl magnetic beads are coupled with the protein antibody, the supernatant is removed by magnetic separation, and then washed successively with a buffer solution and a cleaning agent to obtain immunomagnetic beads;

[0008] (2) The immunomagnetic beads are sprayed on a pretreated nitrocellulose membrane and dried for later use; the diluted antibody solution I and antibody solution II are respectively sprayed on the test line and the quality control line of the nitrocellulose membrane and dried for later use;

[0009] (3) The sample pad, the immunomagnetic bead conjugate pad, the nitrocellulose membrane and the absorbent pad are assembled and cut successively to obtain an immunomagnetic bead test strip, which is stored in a dry and sealed manner.

[0010] The present invention combines magnetic beads with an immunochromatographic test strip, which can not only rapidly enrich proteins in a sample to be tested, but also directly detect whether there are proteins in the sample to be tested on the test strip, avoiding the elution step in the prior art, and at the same time improving the detection sensitivity of the existing test strip. The operation is simple and convenient for rapid detection at the archaeological site.

[0011] Preferably, when the trace protein is fibroin, the protein antibody is a goat anti-rabbit fibroin polyclonal antibody; the antibody solution I is a mouse anti-rabbit fibroin polyclonal antibody solution; the antibody solution II is a goat anti-rabbit antibody solution.

[0012] The goat anti-rabbit fibroin polyclonal antibody uses a silk protein hydrolysate as an antigen, which can stimulate the immune body to produce antibodies with different binding sites. Due to the continuity of the fibroin molecule, it can ensure the continuity of the fibroin antibody, and also ensure that the fibroin antibody has many sites that can bind to the silk protein. The immunomagnetic beads prepared with this antibody also have many sites that can bind to the silk protein, ensuring the enrichment effect of the magnetic beads. The test line antibody is a mouse anti-rabbit fibroin polyclonal antibody, which can effectively capture fibroin. By the double antibody sandwich method, this antibody can also capture the immunomagnetic beads coupled with the fibroin antibody. The control line antibody is a goat anti-rabbit antibody, which only captures the antibody coupled with the magnetic beads. As long as the test strip can be used, the control line will show a color reaction under any circumstances.

[0013] When the trace protein is collagen, the protein antibody is a goat anti-rabbit type I collagen polyclonal antibody; the antibody solution I is a mouse anti-rabbit type I collagen polyclonal antibody solution; the antibody solution II is a goat anti-rabbit antibody solution.

[0014] Preferably, in step (1), the coupling reaction comprises the following steps: adding carboxyl magnetic beads to MES buffer for resuspension, then adding protein antibody and coupling activator, and performing a coupling reaction at room temperature for 2-4 h.

[0015] Preferably, in step (1), the carboxyl magnetic beads have a size of 100-300 nm; the mass ratio of the carboxyl magnetic beads to the protein antibody is 10 mg: 500 μg; the pH of the MES buffer is 5-7; the coupling activator is an EDC·HCl solution with a concentration of 10 mg / mL.

[0016] When the size of the carboxyl magnetic beads is 100-300 nm, it is most suitable. If the size is too small, the chromatography on the nitrocellulose membrane will be too fast, resulting in false positives. If the size is too large, the chromatography on the membrane will not be possible. MES buffers with different pH values will result in different coupling rates of the magnetic beads, and the magnetic beads have the best coupling rate within this range.

[0017] Preferably, in step (1), the buffer is a Tris-HCl solution with a pH of 7.4; the cleaning agent is a PBST solution containing 0.1% (mass fraction) Tween-20 with a pH of 7.4.

[0018] Preferably, in step (1), the step of washing successively with the buffer and the cleaning agent is as follows: adding the carboxyl magnetic beads to the buffer, mixing to resuspend the magnetic beads, rotating and mixing at room temperature for 2-3 h, magnetically separating to remove the supernatant; then adding the cleaning agent and magnetically separating to remove the supernatant.

[0019] Preferably, in step (2), the pretreatment is soaking in MES buffer and drying; the dilution is diluting 500-1000 times with a PBS buffer containing 0.5-2.0% (mass fraction) sucrose.

[0020] When using pure PBS buffer as the diluent for the test strip antibody, the immunomagnetic beads may not be able to chromatograph normally during the chromatography process. Adding sucrose can reduce the diffusion rate of the antibody solution, make the nitrocellulose membrane fully infiltrated, and ensure the smooth chromatography of the immunomagnetic beads.

[0021] Preferably, in step (1), the preparation method of the carboxyl magnetic beads comprises the following steps:

[0022] (a) SiO 2 @Fe 3 O 4 Preparation of magnetic microspheres: Dissolving iron(III) acetylacetonate and 1,12-dodecanediol in phenyl ether, reacting at 180-210 °C for 5-20 min; then adding an ethanol aqueous solution and mixing, and then dropping tetraethyl orthosilicate, heating to 80-100 °C and reacting for 6-10 h. After the reaction is completed, washing and drying to obtain SiO 2@Fe 3 O 4 Magnetic microspheres;

[0023] (b) Preparation of octa-epoxy polyhedral oligomeric silsesquioxane: Mix octa-vinyl polyhedral oligomeric silsesquioxane, concentrated sulfuric acid, 1,3-divinyl-1,1,3,3-tetramethyldisiloxane platinum, and toluene, heat under reflux in an inert atmosphere, dropwise add allyl glycidyl ether and chloroform, and continue heating under reflux for 8 - 12 h; after the reaction is completed, successively carry out vacuum distillation and drying to obtain octa-epoxy polyhedral oligomeric silsesquioxane;

[0024] (c) Preparation of carboxyl magnetic beads: Disperse SiO 2 @Fe 3 O 4 Magnetic microspheres in a mixed solution of octa-epoxy polyhedral oligomeric silsesquioxane, silane coupling agent, and water, stir and reflux for 2 - 4 h, then magnetically attract and wash; disperse the obtained product in a Tris-HCl buffer solution with a pH of 8 - 9, add dopamine and stannous octoate, and carry out a constant temperature water bath reaction at 30 - 40 °C for 6 - 8 h; after magnetic filtration, add carboxymethyl chitosan, glutaraldehyde, glacial acetic acid, and water and mix, react at 60 - 70 °C under stirring for 3 - 5 h, and then successively carry out magnetic attraction, washing, and drying to obtain carboxyl magnetic beads.

[0025] Iron salts will decompose to form Fe 3 O 4 Magnetic nanoparticles, and then tetraethyl orthosilicate is used to coat on their surface to form a core-shell structure of SiO 2 @Fe 3 O 4 Magnetic microspheres, epoxy groups and carboxyl groups are sequentially bonded layer by layer to increase the density of surface functional groups, and finally improve the antibody coupling rate of carboxyl magnetic beads. Octa-epoxy polyhedral oligomeric silsesquioxane has a cage structure with a core composed of silicon-oxygen-silicon bonds (Si-O-Si) and an outer shell of epoxy groups, which has good affinity with the SiO 2 layer on the surface of magnetic microspheres. Moreover, for the carboxyl magnetic beads grafted and branched on the epoxy groups distributed outside the cage structure, the distribution uniformity of carboxyl groups is better, which can improve the effective utilization rate of carboxyl magnetic beads and the enrichment effect of silk fibroin. Dopamine can not only react with epoxy groups by grafting, but also self-polymerize to form a coated layer structure, which can improve the structural stability. At the same time, by controlling the reaction degree, the remaining hydroxyl and amino groups on the outer layer can react with the added carboxymethyl chitosan, and then reactive carboxyl groups are successfully introduced. The carboxyl magnetic beads obtained by layer-by-layer coating and crosslinking have better coating property, better distribution uniformity of surface carboxyl groups, and more uniform magnetic bead particle size distribution, which is beneficial to realizing effective antibody coupling.

[0026] Preferably, in step (a), the molar ratio of ferric acetylacetonate to tetraethyl orthosilicate is 5 - 10:2 - 4.

[0027] Preferably, in step (b), the dosage ratio of octavinyl silsesquioxane, concentrated sulfuric acid, 1,3-divinyl-1,1,3,3-tetramethyldisiloxane platinum, and toluene is 10 - 20 g: 5 - 10 mL: 1 - 3 g: 100 mL; the dosage ratio of octavinyl silsesquioxane, allyl glycidyl ether, and chloroform is 7 - 12 g: 11 - 15 g: 20 - 40 mL.

[0028] Using allyl glycidyl ether for grafting epoxy groups can provide silsesquioxane with flexible alkyl carbon chain segments and extend the spacer arm length between the magnetic beads and surface functional groups, enabling a more effective coupling effect.

[0029] Preferably, in step (c), the mass ratio of SiO 2 @Fe 3 O 4 magnetic microspheres, octaepoxy silsesquioxane, and dopamine is 1: 2 - 3: 2 - 5; the mass ratio of dopamine, carboxymethyl chitosan, and glutaraldehyde is 2 - 5: 1 - 2: 5 - 10; the carboxymethyl substitution degree of carboxymethyl chitosan is not more than 1.

[0030] Since dopamine can also undergo self-polymerization, controlling the mass ratio of raw materials can ensure that there are sufficient cross-linked amino and hydroxyl groups remaining to improve the binding stability. In addition, when the carboxymethyl substitution degree of carboxymethyl chitosan is not more than 1, while ensuring a sufficiently high surface carboxyl density, the reactive functional groups can effectively bind chitosan and magnetic beads.

[0031] In a second aspect, the present invention also provides the application of the immunomagnetic bead test strip prepared by the above preparation method, including the following steps: dissolving the sample to be detected in a protein lysate, taking the supernatant and dropping it on the sample pad of the immunomagnetic bead test strip; after chromatography, observing the color development of the test line and the control line on the immunomagnetic bead test strip; on the premise that the control line shows color, no color on the test line represents negative, and color on the test line represents positive.

[0032] The content of protein degradation products remaining in the imprints, residues, and soil is extremely low, and the molecular weight is small, and it can be dissolved and extracted under mild conditions. After extraction with PBS, the lysate does not need further treatment and can be directly detected on the test paper, which is convenient for on-site archaeological detection.

[0033] Preferably, when the trace protein is silk fibroin, the silk protein lysate is a pH 9.6 carbonate buffer solution or a 50% by mass CaCl 2 solution, the dissolution temperature is 60 - 80 °C, and the time is 45 - 90 min.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] (1) The present invention uses protein antibodies to prepare immunomagnetic beads and combines them with chromatography technology to prepare an immunomagnetic bead chromatography test strip, which can achieve rapid and sensitive detection while enriching trace proteins;

[0036] (2) The carboxyl magnetic beads obtained by layer-by-layer coating and cross-linking have better coating properties, more uniform distribution of surface carboxyl groups, and more uniform particle size distribution of the magnetic beads, which is conducive to realizing effective antibody coupling and improving the antibody coupling rate;

[0037] (3) After the cultural relic sample is dissolved, it can be directly detected on the immunomagnetic bead test strip without further treatment, which is convenient for operation at the archaeological site and takes less time. Description of the Drawings

[0038] Figure 1 Sensitivity detection results of the colloidal gold immunochromatographic test strip in Example 1 (the upper and lower lines are the C line and the T line respectively);

[0039] Figure 2 Sensitivity detection results of the colloidal gold immunochromatographic test strip after immunomagnetic bead enrichment in Example 1;

[0040] Figure 3 Detection results of the textile mineralized cultural relic sample in Example 1;

[0041] Figure 4 Results of influencing the coupling rate in Example 4 and Example 5. Detailed Embodiments

[0042] The present invention will be further described below in conjunction with the embodiments.

[0043] Example 1

[0044] A preparation method of an immunomagnetic bead test strip for rapid detection of trace fibroin includes the following steps:

[0045] (1) Obtaining immunomagnetic beads: Take 1 mL of 100 nm carboxyl magnetic beads (10 mg) and add them to a 2 mL centrifuge tube. Remove the supernatant by magnetic separation; add 400 μL of MES buffer with pH 5.0 and a concentration of 100 mmol / L to resuspend the magnetic beads; add 500 μg of goat anti-rabbit silk fibroin polyclonal antibody, and then add 100 μL of 10 mg / mL coupling activator EDC·HCl solution. Place it on an oscillator for coupling reaction at room temperature for 2 h. After the reaction is completed, collect and remove the supernatant with a magnetic stand; add 1 mL of Tris-HCl buffer with pH 7.4, mix and resuspend the magnetic beads, rotate and mix at room temperature for 2 h, and remove the supernatant by magnetic separation; add 1 mL of PBST (containing 0.1% Tween-20) cleaning agent, and remove the supernatant by magnetic separation to obtain immunomagnetic beads.

[0046] (2) Evaluation of enrichment effect: Prepare silk fibroin standard solutions with concentrations of 1 ng / mL, 10 ng / mL, 100 ng / mL, 1.0 μg / mL, 3.0 μg / mL, and 10 μg / mL using PBS 7.4. As Figure 1 shown, take 500 μL of each silk fibroin standard solution and directly detect it using the test strip developed by Patent CN104459162B. Additionally, take 500 μL of each silk fibroin standard solution and dissolve it in a centrifuge tube, add immunomagnetic beads for enrichment. After 30 min, remove the solution and elute it with methanol. As Figure 2 shown, after drying and re-dissolving, use the test strip developed by Patent CN104459162B to evaluate the enrichment effect of the immunomagnetic beads.

[0047] (3) Preparation of immunomagnetic bead test strip: Immerse the nitrocellulose membrane (UniSart CN140) in MES buffer with pH 5.0. After 30 min, place it in an oven at 37 °C to dry. Spray the prepared immunomagnetic beads evenly on the pretreated nitrocellulose membrane using the airjet nozzle of a film spraying instrument, and dry it at 37 °C for standby. Use a film spraying machine to spray the mouse anti-rabbit silk fibroin polyclonal antibody solution and goat anti-rabbit antibody solution diluted 500 times in PBS buffer containing 0.5% sucrose by mass fraction on the test line (T) and control line (C) of the nitrocellulose membrane with a length of 2.5 cm and a width of 4 mm, ensuring that the distance between the test line and the control line is 10 mm, and dry it at 37 °C for standby. Assemble the sample pad, immunomagnetic bead conjugate pad, nitrocellulose membrane, and absorbent pad on the PVC bottom plate in sequence, cut it into independent test strips with a width of 4 mm using a film cutting machine, and seal and store them with a desiccant.

[0048] Use the immunomagnetic bead test strip prepared above for sample detection:

[0049] Take 1 mg of the mineralized textile sample, dissolve it in 100 μL of carbonate buffer solution with pH 9.6 at 80 °C, stir evenly, and after 90 min, take three drops of the supernatant and drop them on the sample pad of the immunomagnetic bead test strip; after 10 min of chromatography, observe the color development of the T line and C line of the test strip. The identification result of the textile cultural relic in Example 1 is as Figure 3 shown. Both the C line and the T line show color, indicating that the fiber material of the mineralized sample is silk.

[0050] Example 2

[0051] A preparation method of an immunomagnetic bead test strip for rapid detection of trace fibroin, comprising the following steps:

[0052] (1) Obtaining immunomagnetic beads: Take 10 mg of 200 nm carboxyl magnetic beads in 1 mL and add them to a 2 mL centrifuge tube, magnetically separate to remove the supernatant; add 400 μL of MES buffer solution with pH 6.0 and a concentration of 100 mmol / L to resuspend the magnetic beads; add 500 μg of goat anti-rabbit fibroin polyclonal antibody, then add 100 μL of 10 mg / mL coupling activator EDC·HCl solution, and place it on a shaker for coupling reaction at room temperature for 2 h. After the reaction is completed, collect and remove the supernatant with a magnetic rack; add 1 mL of Tris-HCl buffer solution with pH 7.4, mix and resuspend the magnetic beads, rotate and mix at room temperature for 2 h, and magnetically separate to remove the supernatant; add 1 mL of PBST (containing 0.1% Tween-20) cleaning agent with pH 7.4, and magnetically separate to remove the supernatant to obtain immunomagnetic beads.

[0053] (2) Preparation of the immunomagnetic bead test strip: Immerse the nitrocellulose membrane (UniSart CN140) in MES buffer solution with pH 5.0, and after 30 min, place it in an oven at 37 °C to dry. Spray the prepared immunomagnetic beads evenly on the pretreated nitrocellulose membrane with the airjet nozzle of the film spraying instrument, and dry it at 37 °C for standby. Spray the mouse anti-rabbit fibroin polyclonal antibody solution and the goat anti-rabbit antibody solution diluted 500 times in PBS buffer solution containing 1.0% sucrose by mass fraction on the detection line (T) and the quality control line (C) of the nitrocellulose membrane with a length of 2.5 cm and a width of 4 mm respectively, ensure that the distance between the detection line and the quality control line is 10 mm, and dry it at 37 °C for standby. Assemble the sample pad, the immunomagnetic bead binding pad, the nitrocellulose membrane and the absorbent pad on the PVC bottom plate in sequence, cut it into independent test strips with a width of 4 mm with a film cutting machine, and seal and store them with a desiccant.

[0054] Use the above-prepared immunomagnetic bead test strip for sample detection:

[0055] Take 2 mg of the textile imprint sample and dissolve it in 1 mL of pH 9.6 carbonate buffer solution at 70 °C. Stir evenly. After 60 min, take three drops of the supernatant and drop them on the sample pad of the immunomagnetic bead test strip. After 10 min of chromatography, observe the color development of the T line and C line on the test strip. The identification result of the textile cultural relic in Example 1 was that both the C line and the T line showed color, indicating that the fiber material of the imprint sample was silk.

[0056] Example 3

[0057] A preparation method of an immunomagnetic bead test strip for rapid detection of trace fibroin, comprising the following steps:

[0058] (1) Obtaining immunomagnetic beads: Take 10 mg of 300 nm carboxyl magnetic beads in 1 mL and add them to a 2 mL centrifuge tube. Magnetically separate to remove the supernatant. Add 400 μL of MES buffer solution with pH 7.0 and a concentration of 100 mmol / L to resuspend the magnetic beads. Add 500 μg of goat anti-rabbit fibroin polyclonal antibody, and then add 100 μL of 10 mg / mL coupling activator EDC·HCl solution. Place it on an oscillator and perform a coupling reaction at room temperature for 2 h. After the reaction is completed, use a magnetic rack to collect and remove the supernatant. Add 1 mL of Tris-HCl buffer solution with pH 7.4, mix and resuspend the magnetic beads, rotate and mix at room temperature for 2 h, and magnetically separate to remove the supernatant. Add 1 mL of PBST (containing 0.1% Tween-20) cleaning agent, and magnetically separate to remove the supernatant to obtain immunomagnetic beads.

[0059] (2) Preparation of the immunomagnetic bead test strip: Immerse the nitrocellulose membrane (UniSart CN140) in MES buffer solution with pH 5.0. After 30 min, place it in an oven at 37 °C and dry it. Spray the prepared immunomagnetic beads evenly on the pretreated nitrocellulose membrane with the airjet nozzle of the film spraying instrument, and dry it at 37 °C for standby. Use a film spraying machine to spray the mouse anti-rabbit fibroin polyclonal antibody solution and the goat anti-rabbit antibody solution diluted 1000 times in PBS buffer solution containing 1.5% sucrose by mass fraction on the detection line (T) and the quality control line (C) of the nitrocellulose membrane with a length of 2.5 cm and a width of 4 mm respectively, ensuring that the distance between the detection line and the quality control line is 10 mm, and dry it at 37 °C for standby. Assemble the sample pad, the immunomagnetic bead binding pad, the nitrocellulose membrane and the absorbent pad on the PVC bottom plate in sequence, cut them into independent test strips with a width of 4 mm with a film cutting machine, and seal them with a desiccant for storage.

[0060] Use the above-prepared immunomagnetic bead test strip for sample detection:

[0061] Take 10 mg of soil sample, dissolve it in 1 mL of carbonate buffer solution with pH 9.6 at 70 °C, stir evenly, and take three drops of the supernatant after 45 min and drop them on the sample pad of the immunomagnetic bead test strip; after 10 min of chromatography, observe the color development of the T line and C line on the test strip. The identification result of the textile cultural relic in Example 1 finally shows that both the C line and the T line are colored, indicating that the soil sample contains silk.

[0062] Example 4

[0063] Detect the influence of different contents of goat anti-rabbit silk fibroin polyclonal antibody on the coupling rate of carboxyl magnetic beads:

[0064] Take 10 mg of 1 mL of 100 nm carboxyl magnetic beads and add them to a 2 mL centrifuge tube, remove the supernatant by magnetic separation; add 400 μL of MES buffer solution with pH 5.0 and a concentration of 100 mmol / L to resuspend the magnetic beads; add goat anti-rabbit silk fibroin polyclonal antibody, and set 8 parallel groups according to different contents of goat anti-rabbit silk fibroin polyclonal antibody, so that the antibody content (antibody addition amount / carboxyl magnetic bead addition amount) is 30 μg / mg, 40 μg / mg, 50 μg / mg, 60 μg / mg, 70 μg / mg, 80 μg / mg, 90 μg / mg, 100 μg / mg respectively; then add 100 μL of 10 mg / mL coupling activator EDC·HCl solution, and place it on a shaker for coupling reaction at room temperature for 2 h. After the reaction is completed, collect the carboxyl magnetic beads with a magnetic stand and calculate the coupling rate.

[0065] Example 5

[0066] Detect the influence of MES buffer solution with different pH values on the coupling rate of carboxyl magnetic beads:

[0067] Take 10 mg of 1 mL of 100 nm carboxyl magnetic beads and add them to a 2 mL centrifuge tube, remove the supernatant by magnetic separation; add 400 μL of MES buffer solution with a concentration of 100 mmol / L to resuspend the magnetic beads, and set 5 parallel groups with MES buffer solutions of different pH values respectively, and the pH values are 5.0, 5.5, 6.0, 6.5, 7.0; add 500 μg of goat anti-rabbit silk fibroin polyclonal antibody, then add 100 μL of 10 mg / mL coupling activator EDC·HCl solution, and place it on a shaker for coupling reaction at room temperature for 2 h. After the reaction is completed, collect the carboxyl magnetic beads with a magnetic stand and calculate the coupling rate.

[0068] Example 6

[0069] A preparation method of an immunomagnetic bead test strip for rapid detection of trace collagen, comprising the following steps:

[0070] (1) Obtaining immunomagnetic beads: Take 1 mL of 100 nm carboxyl magnetic beads (10 mg) and add them to a 2 mL centrifuge tube. Remove the supernatant by magnetic separation; add 400 μL of MES buffer with pH 5.0 and a concentration of 100 mmol / L to resuspend the magnetic beads; add 500 μg of goat anti-rabbit type I collagen polyclonal antibody, and then add 100 μL of 10 mg / mL coupling activator EDC·HCl solution. Place it on an oscillator for coupling reaction at room temperature for 3 h. After the reaction is completed, collect and remove the supernatant with a magnetic stand; add 1 mL of Tris-HCl buffer with pH 7.4, mix and resuspend the magnetic beads, rotate and mix at room temperature for 4 h, and remove the supernatant by magnetic separation; add 1 mL of PBST (containing 0.1% Tween-20) cleaning agent, and remove the supernatant by magnetic separation to obtain immunomagnetic beads.

[0071] (2) Preparation of immunomagnetic bead test strips: Immerse the nitrocellulose membrane (UniSart CN140) in MES buffer with pH 5.0, and place it in an oven at 37 °C to dry after 30 min. Spray the prepared immunomagnetic beads evenly on the pretreated nitrocellulose membrane with the airjet nozzle of a film spraying instrument, and dry it at 37 °C for standby. Use a film spraying machine to spray the mouse anti-rabbit type I collagen polyclonal antibody solution and goat anti-rabbit antibody solution diluted 500 times in PBS buffer containing 0.5% sucrose by mass fraction on the test line (T) and control line (C) of the nitrocellulose membrane with a length of 2.5 cm and a width of 4 mm respectively, ensuring that the distance between the test line and the control line is 10 mm, and dry it at 37 °C for standby. Assemble the sample pad, immunomagnetic bead conjugate pad, nitrocellulose membrane and absorbent pad on the PVC bottom plate in sequence, cut it into independent test strips with a width of 4 mm with a film cutting machine, and seal it with a desiccant for storage.

[0072] Use the above-prepared immunomagnetic bead test strips for sample detection:

[0073] Take 1 mg of the fiber residue sample from the archaeological site, dissolve it in 100 μL of carbonate buffer with pH 9.6 at 80 °C, stir evenly, and take three drops of the supernatant after 120 min and drop them on the sample pad of the immunomagnetic bead test strip; after chromatography for 20 min, observe the color development of the T line and C line on the test strip. If both the C line and the T line show color, it indicates that the fiber residue sample contains collagen and the fiber is leather collagen fiber.

[0074] Example 7

[0075] The preparation method of carboxyl magnetic beads includes the following steps:

[0076] (a) SiO 2 @Fe 3 O 4Preparation of magnetic microspheres: Dissolve 2.47 g of iron(III) acetylacetonate (353.17) and 7.08 g of 1,12-dodecanediol (202.33, 5) in 50 mL of phenyl ether, and react at 190 °C for 15 min; then add 100 mL of an ethanol aqueous solution (volume ratio 1:1) and mix. After that, add 0.62 g of tetraethyl orthosilicate (208.33), heat up to 85 °C and react for 8 h. After the reaction is completed, wash and dry to obtain SiO 2 @Fe 3 O 4 magnetic microspheres;

[0077] (b) Preparation of octaepoxy polyhedral oligomeric silsesquioxane: Add 12 g of octavinyl polyhedral oligomeric silsesquioxane, 8 mL of concentrated sulfuric acid, and 1.5 g of 1,3-divinyl-1,1,3,3-tetramethyldisiloxane platinum to 100 mL of toluene and mix. Heat under reflux in a nitrogen atmosphere, then dropwise add a 30 mL chloroform solution containing 12 g of allyl glycidyl ether, and continue to heat under reflux for 8 - 12 h; after the reaction is completed, successively perform vacuum distillation and drying to obtain octaepoxy polyhedral oligomeric silsesquioxane;

[0078] (c) Preparation of carboxyl magnetic beads: Disperse 1 g of SiO 2 @Fe 3 O 4 magnetic microspheres in a mixed solution of 2.5 g of octaepoxy polyhedral oligomeric silsesquioxane, 0.5 g of silane coupling agent, and 150 mL of water, stir and reflux for 3 h, then perform magnetic separation and washing; disperse the obtained product in 500 mL of Tris-HCl buffer solution with a pH of 8.5, add 2 g of dopamine and 0.2 g of stannous octoate, react in a constant temperature water bath at 35 °C for 6 h, perform magnetic separation and filtration, then add 1.2 g of carboxymethyl chitosan (carboxymethyl substitution degree 0.90), 6 g of glutaraldehyde, 10 mL of glacial acetic acid, and 60 mL of water and mix, react at 65 °C under stirring for 4 h, and then successively perform magnetic separation, washing, and drying to obtain carboxyl magnetic beads.

[0079] Take 10 mg of the carboxyl magnetic beads prepared above and add them to a 2 mL centrifuge tube, perform magnetic separation to remove the supernatant; add 400 μL of MES buffer solution with a pH of 6.0 and a concentration of 100 mmol / L to resuspend the magnetic beads; add 700 μg of goat anti-rabbit silk fibroin polyclonal antibody, then add 100 μL of 10 mg / mL coupling activator EDC·HCl solution, and place it on an oscillator for room temperature coupling reaction for 2 h. After the reaction is completed, collect the carboxyl magnetic beads with a magnetic stand and calculate the coupling rate.

[0080] Example 8

[0081] The preparation method of carboxyl magnetic beads includes the following steps:

[0082] (a) SiO2 @Fe 3 O 4 Preparation of magnetic microspheres: Dissolve 2.47 g of iron(III) acetylacetonate (353.17) and 7.08 g of 1,12-dodecanediol (202.33, 5) in 50 mL of phenyl ether, and react at 190 °C for 15 min; then add 100 mL of an ethanol aqueous solution (volume ratio 1:1) and mix. After that, add 0.62 g of tetraethyl orthosilicate (208.33), and raise the temperature to 85 °C and react for 8 h. After the reaction is completed, wash and dry to obtain SiO 2 @Fe 3 O 4 magnetic microspheres;

[0083] (b) Preparation of octaepoxycaged silsesquioxane: Add 12 g of octavinylcaged silsesquioxane, 8 mL of concentrated sulfuric acid, and 1.5 g of 1,3-divinyl-1,1,3,3-tetramethyldisiloxane platinum to 100 mL of toluene and mix. Heat under reflux in a nitrogen atmosphere, and then dropwise add a 30 mL chloroform solution containing 12 g of allyl glycidyl ether, and continue to heat under reflux for 8 - 12 h; after the reaction is completed, successively perform vacuum distillation and drying to obtain octaepoxycaged silsesquioxane;

[0084] (c) Preparation of carboxyl magnetic beads: Disperse 1 g of SiO 2 @Fe 3 O 4 magnetic microspheres in a mixed solution of 2.2 g of octaepoxycaged silsesquioxane, 0.5 g of silane coupling agent, and 150 mL of water, stir and reflux for 3 h, and then perform magnetic separation and washing; disperse the obtained product in 600 mL of Tris-HCl buffer solution with a pH of 8.5, add 3.5 g of dopamine and 0.3 g of stannous octoate, and react in a constant temperature water bath at 35 °C for 7 h. After magnetic filtration, add 1.5 g of carboxymethyl chitosan (carboxymethyl substitution degree 0.90), 7 g of glutaraldehyde, 10 mL of glacial acetic acid, and 60 mL of water and mix, and react at 60 °C for 5 h under stirring conditions. Then, successively perform magnetic separation, washing, and drying to obtain carboxyl magnetic beads.

[0085] Take 10 mg of the carboxyl magnetic beads prepared above and add them to a 2 mL centrifuge tube, and magnetically separate to remove the supernatant; add 400 μL of MES buffer solution with a pH of 6.0 and a concentration of 100 mmol / L to resuspend the magnetic beads; add 700 μg of goat anti-rabbit silk fibroin polyclonal antibody, and then add 100 μL of 10 mg / mL coupling activator EDC·HCl solution, and place it on an oscillator to couple and react at room temperature for 2 h. After the reaction is completed, collect the carboxyl magnetic beads with a magnetic stand and calculate the coupling rate.

[0086] Comparative Example 1

[0087] The difference from Example 7 is as follows: MPS100 / carboxyl magnetic beads from Shanghai Yinruicheng Biotechnology Co., Ltd. were used. Take 10 mg of commercially available carboxyl magnetic beads and add them to a 2 mL centrifuge tube. Remove the supernatant by magnetic separation; add 400 μL of MES buffer solution with a pH of 6.0 and a concentration of 100 mmol / L to resuspend the magnetic beads; add 700 μg of goat anti-rabbit silk fibroin polyclonal antibody, and then add 100 μL of 10 mg / mL coupling activator EDC·HCl solution. Place it on an oscillator and perform a coupling reaction at room temperature for 2 h. After the reaction is completed, collect the carboxyl magnetic beads with a magnetic rack and calculate the coupling rate.

[0088] Comparative Example 2

[0089] The difference from Example 7 is as follows: The carboxymethyl substitution degree of carboxymethyl chitosan is 1.1.

[0090] The preparation method of the carboxyl magnetic beads includes the following steps:

[0091] (a) Preparation of SiO 2 @Fe 3 O 4 Magnetic microspheres: Dissolve 2.47 g of iron(III) acetylacetonate (353.17) and 7.08 g of 1,12-dodecanediol (202.33, 5) in 50 mL of phenyl ether, and react at 190 °C for 15 min; then add 100 mL of ethanol aqueous solution (volume ratio 1:1) and mix. After that, add 0.62 g of tetraethyl orthosilicate (208.33) dropwise and heat to 85 °C for 8 h. After the reaction is completed, wash and dry to obtain SiO 2 @Fe 3 O 4 Magnetic microspheres;

[0092] (b) Preparation of octa(epoxypropyl)silsesquioxane: Add 12 g of octavinylsilsesquioxane, 8 mL of concentrated sulfuric acid, and 1.5 g of 1,3-divinyl-1,1,3,3-tetramethyldisiloxane platinum to 100 mL of toluene and mix. Heat under reflux in a nitrogen atmosphere, and then add dropwise a 30 mL chloroform solution containing 12 g of allyl glycidyl ether, and continue to heat under reflux for 8 - 12 h; after the reaction is completed, perform vacuum distillation and drying in sequence to obtain octa(epoxypropyl)silsesquioxane;

[0093] (c) Preparation of carboxyl magnetic beads: Add 1 g of SiO 2 @Fe 3 O 4The magnetic microspheres were dispersed in a mixed solution of 2.5 g of octaepoxycage silsesquioxane, 0.5 g of silane coupling agent and 150 mL of water, and stirred and refluxed for 3 h, then magnetically separated and washed; the obtained product was dispersed in 500 mL of Tris-HCl buffer solution with a pH of 8.5, 2 g of dopamine and 0.2 g of stannous octoate were added, and the reaction was carried out in a constant temperature water bath at 35 °C for 6 h. After magnetic separation and filtration, 1.2 g of carboxymethyl chitosan (carboxymethyl substitution degree 1.1), 6 g of glutaraldehyde, 10 mL of glacial acetic acid and 60 mL of water were added and mixed, and the reaction was carried out at 65 °C for 4 h under stirring conditions. Then, it was successively subjected to magnetic separation, washing and drying to obtain carboxyl magnetic beads.

[0094] Take 10 mg of the carboxyl magnetic beads prepared above and add them to a 2 mL centrifuge tube, magnetically separate to remove the supernatant; add 400 μL of MES buffer solution with a pH of 6.0 and a concentration of 100 mmol / L to resuspend the magnetic beads; add 700 μg of goat anti-rabbit silk fibroin polyclonal antibody, and then add 100 μL of 10 mg / mL coupling activator EDC·HCl solution, and place it on an oscillator for room temperature coupling reaction for 2 h. After the reaction is completed, collect the carboxyl magnetic beads with a magnetic stand and calculate the coupling rate.

[0095] Table 1

[0096] Example 7 Example 8 Comparative Example 1 Comparative Example 2 OD value of the previous concentration 1.532 1.533 1.535 1.530 OD value of the subsequent concentration 0.428 0.444 0.537 0.490 Coupling rate (%) 72.06 71.04 65.02 67.97

[0097] The solutions before and after antibody coupling were detected by BCA, the absorbance values were substituted into the standard curve to calculate the concentration, and finally the coupling rate was calculated. The coupling rate (%) = (concentration before coupling - concentration after coupling) / concentration before coupling.

[0098] The present invention uses silk fibroin polyclonal antibody to prepare immunomagnetic beads and combines them with chromatography technology to prepare an immunomagnetic bead chromatography test strip. Figure 1-2 It shows that the immunomagnetic beads have a good enrichment effect on silk fibroin, while Figure 3 it shows that it can achieve rapid and sensitive detection while enriching trace silk fibroin. As Figure 4 shown, Figure (a) shows the results of the influence of different contents of goat anti-rabbit silk fibroin polyclonal antibody on the coupling rate. The results show that when the antibody content is 70 μg / mg, the coupling rate of carboxyl magnetic beads is the highest. Figure (b) shows the results of the influence of MES buffer solutions with different pH values on the coupling rate. The results show that when the pH value of the MES buffer solution is 6.0, the coupling rate of carboxyl magnetic beads is the highest.

[0099] In the present invention, the carboxyl magnetic beads prepared are subjected to a coupling reaction when the antibody content is 70 μg / mg and the pH value of the MES buffer solution is 6.0. It can be seen from Example 7 and Comparative Example 1 that the coupling rate thereof is higher than that of the commercially available carboxyl magnetic beads, and the coupling effect is better, so that a more sensitive detection of trace fibroin can be achieved. It can be seen from Example 7 and Comparative Example 2 that when the carboxymethyl substitution degree of carboxymethyl chitosan is greater than 1, carboxymethyl substitution will occur on the amino group at the same time, which will reduce the reactive functional groups effectively combined by chitosan and magnetic beads, and is instead not conducive to increasing the carboxyl density on the surface of the magnetic beads.

[0100] Unless otherwise specified, the raw materials and equipment used in the present invention are all common raw materials and equipment in the art; unless otherwise specified, the methods used in the present invention are all conventional methods in the art.

[0101] The above are only the preferred embodiments of the present invention, and do not impose any limitation 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 an immunomagnetic bead test strip for rapid detection of trace proteins in cultural relics, characterized in that, it includes: (a) Preparation of SiO 2 @ Fe 3 O 4 magnetic microspheres; (b) Mix octavinylcage silsesquioxane, concentrated sulfuric acid, 1,3-divinyl-1,1,3,3-tetramethyldisiloxane platinum, and toluene, heat under reflux in an inert atmosphere, add allyl glycidyl ether and chloroform dropwise, and continue heating under reflux; After vacuum distillation and drying, octaepoxycage silsesquioxane is obtained; (c) Disperse SiO 2 @Fe 3 O 4 magnetic microspheres in a mixed solution of octa(epoxypropyl)silsesquioxane, silane coupling agent and water, stir and reflux for 2 - 4 h, magnetically separate and wash; disperse the obtained product in a Tris-HCl buffer solution with a pH of 8 - 9, add dopamine and stannous octoate, react at 30 - 40 °C for 6 - 8 h; after magnetic filtration, add carboxymethyl chitosan with a carboxymethyl substitution degree not greater than 1, glutaraldehyde, glacial acetic acid and water and mix, stir and react at 60 - 70 °C for 3 - 5 h, and obtain carboxyl magnetic beads by magnetic separation; (1) After the carboxyl magnetic beads are subjected to a coupling reaction with the protein antibody, the supernatant is removed by magnetic separation, and after washing with a buffer solution and a cleaning agent in sequence, immunomagnetic beads are obtained; (2) Spray the immunomagnetic beads on a pretreated nitrocellulose membrane and dry; spray the diluted antibody solution I and antibody solution II on the test line and the quality control line of the nitrocellulose membrane respectively and dry; (3) Assemble and cut the sample pad, immunomagnetic bead conjugate pad, nitrocellulose membrane, and absorbent pad in sequence.

2. The method for preparing an immunomagnetic bead test strip for rapid detection of trace proteins in cultural relics according to claim 1, characterized in that, when the trace protein is fibroin, the protein antibody is a goat anti-rabbit fibroin polyclonal antibody; the antibody solution I is a mouse anti-rabbit fibroin polyclonal antibody solution; the antibody solution II is a goat anti-rabbit antibody solution; when the trace protein is collagen, the protein antibody is a goat anti-rabbit type I collagen polyclonal antibody; the antibody solution I is a mouse anti-rabbit type I collagen polyclonal antibody solution; the antibody solution II is a goat anti-rabbit antibody solution.

3. The method for preparing an immunomagnetic bead test strip for rapid detection of trace proteins in cultural relics according to claim 1, characterized in that, in step (1), the coupling reaction includes the following steps: resuspend the carboxyl magnetic beads in MES buffer solution, then add the protein antibody and a coupling activator, and carry out a room temperature coupling reaction for 2-4 h.

4. The method for preparing an immunomagnetic bead test strip for rapid detection of trace proteins in cultural relics according to claim 3, characterized in that, in step (1), the size of the carboxyl magnetic beads is 100-300 nm; the mass ratio of the carboxyl magnetic beads to the protein antibody is 10 mg: 500 μg; the pH of the MES buffer solution is 5-7; the coupling activator is an EDC·HCl solution with a concentration of 10 mg / mL.

5. The method for preparing an immunomagnetic bead test strip for rapid detection of trace proteins in cultural relics according to claim 1, characterized in that, in step (1), the buffer solution is a Tris-HCl solution with a pH of 7.4; the cleaning agent is a PBST solution containing 0.1% Tween-20 by mass fraction and a pH of 7.

4.

6. The method for preparing an immunomagnetic bead test strip for rapid detection of trace proteins in cultural relics according to claim 5, characterized in that, in step (1), the step of washing with the buffer solution and the cleaning agent in sequence is: add the carboxyl magnetic beads to the buffer solution, mix and resuspend the magnetic beads, rotate and mix at room temperature for 2-3 h, and remove the supernatant by magnetic separation; after adding the cleaning agent, remove the supernatant by magnetic separation.

7. The method for preparing an immunomagnetic bead test strip for rapid detection of trace proteins in cultural relics according to claim 1, It is characterized in that In step (2), the pretreatment is soaking in MES buffer solution and drying; the dilution is diluting 500 - 1000 times with PBS buffer solution containing 0.5 - 2.0% sucrose by mass fraction.

8. The preparation method of the immunomagnetic bead test strip for rapid detection of trace proteins in cultural relics as claimed in claim 1 It is characterized in that In step (1), the SiO 2 @Fe 3 O 4 The method for preparing magnetic microspheres comprises the following steps: Dissolve iron(III) acetylacetonate and 1,12-dodecanediol in diphenyl ether, and react at 180 - 210 °C for 5 - 20 min; then add an ethanol aqueous solution and mix, and then dropwise add tetraethyl orthosilicate, raise the temperature to 80 - 100 °C and react for 6 - 10 h. After the reaction is completed, wash and dry to obtain SiO 2 @ Fe 3 O 4 magnetic microspheres.

9. The preparation method of the immunomagnetic bead test strip for rapid detection of trace proteins in cultural relics as claimed in claim 8 It is characterized in that In step (a), the molar ratio of iron(III) acetylacetonate and tetraethyl orthosilicate is 5 - 10:2 - 4; In step (b), the dosage ratio of octavinylsilsesquioxane, concentrated sulfuric acid, 1,3 - divinyl - 1,1,3,3 - tetramethyldisiloxane platinum, and toluene is 10 - 20 g:5 - 10 mL:1 - 3 g:100 mL; the dosage ratio of octavinylsilsesquioxane, allyl glycidyl ether, and chloroform is 7 - 12 g:11 - 15 g:20 - 40 mL; In step (c), the mass ratio of SiO 2 @ Fe 3 O 4 magnetic microspheres, octaepoxycage silsesquioxane and dopamine is 1: 2-3: 2-5; the mass ratio of dopamine, carboxymethyl chitosan and glutaraldehyde is 2-5: 1-2: 5-10.

10. The application of the immunomagnetic bead test strip prepared by the preparation method as claimed in any one of claims 1 - 9 It is characterized in that It includes the following steps: dissolving the sample to be detected in a protein dissolution solution, taking the supernatant and dropping it on the sample pad of the immunomagnetic bead test strip; after chromatography, observing the color development of the test line and the control line on the immunomagnetic bead test strip; on the premise that the control line shows color, no color on the test line represents negative, and color on the test line represents positive.

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