A method for immunomagnetic enhanced detection of sheep leather

Abstract

The present invention relates to the field of cultural relics detection, and discloses a method for immunomagnetic enhanced detection of sheep leather, comprising the preparation of nano CuO, nano immunomagnetic beads, functionalized nano magnetic bead microparticles, specific magnetic beads, specific CuO, and adding specific magnetic beads and specific CuO to a sheep leather protein solution to be detected, shaking and incubating, removing the supernatant after centrifugation, placing in hydrogen peroxide, and passing the obtained oxygen into a pressure sensor. The present invention combines antigen antibodies with a pressure sensor to provide a sheep leather protein detection method with the advantages of immunospecificity, which has stronger specificity and lower detection limit.

Description

Technical Field

[0001] The invention relates to the field of cultural relics detection, in particular to a method for immunomagnetic enhancement detection of sheepskin. Background Art

[0002] my country is one of the earliest countries to use textiles. These textiles have witnessed the social changes, economic development and cultural integration of my country, and are precious historical materials for studying the level of social, economic, technological and cultural level in ancient my country. Most of the textile cultural relics currently released are protein cultural relics, among which leather textiles are easily affected by moisture, temperature, pH and microorganisms in the tomb environment, causing aging and decomposition, and serious damage. For the identification of leather cultural relics, finding a highly sensitive, specific, simple, fast and efficient species identification method is of great significance to the study of the origin of my country's leather cultural relics, and at the same time provides a new scientific basis for the study of China's thousands of years of clothing culture. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a method for immunomagnetic enhancement detection of sheep leather.

[0004] The specific technical solution of the present invention is: a method for immunomagnetic enhanced detection of sheep leather, comprising the following steps:

[0005] Step 1: Preparation of nano CuO: Mix copper nitrate solution and sodium carbonate solution to obtain Cu, disperse by ultrasonication, react at 160-200°C for 4-8h, filter under vacuum, wash the obtained powder with water and anhydrous ethanol, and finally dry to obtain nano CuO.

[0006] Step 2: Preparation of nano-immunomagnetic beads: add ferrous oxalate, dodecanediol, dextran and oleic acid into 25 mL of ethanol to dissolve to obtain a mixed solution; heat the obtained mixed solution for reaction and then cool it to obtain a cooling solution containing a reaction product; wash the reaction product in the cooling solution to obtain nano-immunomagnetic beads.

[0007] Step 3: Take the nano-immunomagnetic beads, add water, and disperse them evenly by ultrasonication. Then add chloroform, anhydrous ethanol, and then add acrylamide, acetonitrile and methacrylic acid. Stir rapidly at room temperature. After fully mixing, add the initiator azobisisobutyronitrile, heat and stir rapidly to react, use an external magnetic field for solid-liquid separation to obtain functionalized nano-magnetic beads, and wash repeatedly with anhydrous ethanol and water until excess organic matter is washed away.

[0008] In the present invention, acrylamide, acetonitrile and methacrylic acid are added in step 3 as organic synthesis raw materials, so that the magnetic beads have flocculation properties for proteins, which can further enhance the ability of the magnetic beads to bind proteins and reduce the detection limit, so that even trace amounts of protein can be detected.

[0009] Step 4: Preparation of specific magnetic beads: add functionalized nano magnetic beads to disodium hydrogen phosphate-sodium dihydrogen phosphate buffer and disperse by ultrasonic; add glutaraldehyde solution and react in a water bath; magnetically separate the solid and liquid phases, wash with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, disperse the activated magnetic beads in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to obtain an activated magnetic bead solution, add sheep leather protein antibody to the activated magnetic bead solution and shake, add BSA after washing and mix well to obtain specific magnetic beads after washing.

[0010] Step 5: Preparation of specific CuO: Take nano-CuO and ultrasonicate it in PBS, add sheep leather protein antibody to the obtained dispersion and shake it; remove the upper clear liquid after freeze centrifugation, disperse it in PBS, remove the lower substrate after freeze centrifugation again, add BSA solution, mix and react, and obtain specific CuO.

[0011] Step 6: Add the obtained specific magnetic beads and specific CuO to the sheep leather protein solution to be tested, incubate with shaking, remove the supernatant after centrifugation, place it in hydrogen peroxide, and pass the obtained oxygen into the pressure sensor to cause the pressure change to cause the resistance change to obtain the wool leather protein concentration.

[0012] Preferably, step 1 is specifically as follows: 25-35 mL 3-7 mg / mL copper nitrate solution and 25-35 mL 8-12 mg / mL sodium carbonate solution are mixed and ultrasonically dispersed for 0.5-1 h, reacted at 160-200° C. for 4-8 h, vacuum filtered, and the obtained powder is washed with water and anhydrous ethanol successively, and finally dried at 80-100° C. for 1.5-2 h to obtain nano CuO.

[0013] Preferably, step 2 is specifically as follows: 1.5-2.5 mmol ferric oxalate, 8-12 mol dodecanediol, 0.6-1.0 g dextran and 4-8 mol oleic acid are added to 20-30 mL ethanol to dissolve to obtain a mixed solution; the obtained mixed solution is reacted at 180-250° C. for 5-30 min and then cooled to obtain a cooling solution containing a reaction product; the reaction product in the cooling solution is washed to obtain immunomagnetic beads. Preferably, step 3 is specifically as follows: take 0.3-0.5g of nano-immunomagnetic beads, add 20mL of water, ultrasonically disperse evenly, then add 8-12mL of chloroform, 2-3mL of anhydrous ethanol, then add 0.3-0.7g of acrylamide, 0.8-1.2mL of acetonitrile and 2-3mL of methacrylic acid, stir rapidly for 10-30min at room temperature, after fully mixing, add 0.08-0.12g of initiator azobisisobutyronitrile, stir rapidly at 40-50°C for 6-8h, use an external magnetic field for solid-liquid separation to obtain functionalized nano-magnetic beads, and wash repeatedly with anhydrous ethanol and water until excess organic matter is washed away.

[0014] Preferably, step 4 is specifically as follows: 400-600 mg of functionalized nano-magnetic beads are added to 80-120 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and ultrasonically dispersed; 150-250 mL of 20-30 wt% glutaraldehyde solution is added, and the reaction is oscillated in a water bath at 30-40° C. for 2-3 hours; the solid-liquid phase is magnetically separated, and the activated magnetic beads are dispersed in 40-60 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to obtain 8-12 mg / mL of activated magnetic bead solution, 350-450 uL of the activated magnetic bead solution is taken, 80-120 uL of sheep leather protein antibody solution is added and shaken, and after washing, 150-250 uL of 2-4% BSA solution is added to mix and react, and specific magnetic beads are obtained after washing.

[0015] Preferably, step 5 is specifically as follows: 0.8-1.2 mg of nano-CuO is taken in 0.8-1.2 mL of PBS and ultrasonicated for 0.5-1.5 h, 80-120 uL of sheep leather protein antibody is added to the obtained dispersion, and the dispersion is shaken at 500-700 rpm for 3-5 h; after freeze centrifugation, the upper clear liquid is removed, and the dispersion is dispersed in 0.8-1.2 mL of PBS, and after freeze centrifugation, the lower substrate is removed, 350-450 uL of 2-4% BSA solution is added, and the mixture is mixed and reacted for 20-40 min to obtain specific CuO.

[0016] Preferably, in step 5, the first centrifugation rate is 8000-12000 rpm, and the time is 10-20 min; the second centrifugation rate is 800-1200 rpm, and the time is 5-15 min.

[0017] Preferably, step 6 is as follows: adding the obtained specific magnetic beads and specific nano-CuO to the sheep leather protein solution to be detected, incubating with shaking at 30-40°C for 1-3h, removing the supernatant after centrifugation, placing it in hydrogen peroxide, and passing the obtained oxygen into the pressure sensor.

[0018] Compared with the prior art, the present invention has the following technical effects:

[0019] (1) Due to the specific binding of antigen and antibody, the present invention can eliminate the interference of various impurities, reduce the detection limit, and improve the detection specificity and accuracy.

[0020] (2) The present invention utilizes the principle of CuO catalyzing hydrogen peroxide to significantly improve detection efficiency.

[0021] (3) The present invention utilizes the paramagnetism of magnetic beads to better separate the synthesized materials from the reaction system, thereby facilitating the washing and purification of the synthesized materials. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with the embodiments.

[0023] Example 1

[0024] A method for immunomagnetic enhancement detection of sheep leather comprises the following steps:

[0025] Step 1: Mix 30 mL of 5 mg / mL copper nitrate solution and 30 mL of 10 mg / mL sodium carbonate solution, and disperse them by ultrasonication for 0.5 h. After reacting at 160 ° C for 4 h, vacuum filter the resulting powder, wash it with water and anhydrous ethanol, and finally dry it at 80 ° C for 1.5 h to obtain nano CuO.

[0026] Step 2: Add 2 mmol of ferric oxalate, 10 mol of dodecanediol, 0.8 g of dextran and 6 mol of oleic acid into 25 mL of ethanol to dissolve and obtain a mixed solution; react the obtained mixed solution at 220° C. for 20 min and then cool it to obtain a cooling solution containing a reaction product; wash the reaction product in the cooling solution to obtain immunomagnetic beads.

[0027] Step 3: Take 0.4g of nano-immunomagnetic beads, add 20mL of water, and disperse evenly by ultrasonication. Then add 10mL of chloroform, 2.5mL of anhydrous ethanol, and then add 0.5g of acrylamide, 1.0mL of acetonitrile and 2.5mL of methacrylic acid. Stir rapidly for 20min at room temperature. After fully mixing, add 0.1g of initiator azobisisobutyronitrile, and stir rapidly at 45°C for 7h. Use an external magnetic field for solid-liquid separation to obtain functionalized nano-magnetic beads, and wash repeatedly with anhydrous ethanol and water until excess organic matter is washed away.

[0028] Step 4: Take 500 mg of functionalized nano-magnetic beads, add them to 100 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4, and disperse them by ultrasonic dispersion; add 200 mL of 23 wt% glutaraldehyde solution, and react in a water bath at 37°C for 2.5 h; magnetically separate the solid and liquid phases, wash them 5 times with PBS at pH 7.4, and disperse the activated magnetic beads in 50 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 to obtain a 10 mg / mL activated magnetic bead solution. Take 400 uL of the activated magnetic bead solution, add 100 uL of sheep leather protein antibody solution and shake it, add 200 uL of 3% BSA solution after washing, mix and react, and obtain specific magnetic beads after washing, which are dispersed in 200 uL of phosphate buffer.

[0029] Step 5: Take 1.0 mg of nano-CuO and ultrasonicate it in 1.0 mL of PBS for 1 hour. Add 100 uL of sheep leather protein antibody to the obtained dispersion and shake it at 600 rpm for 4 hours. After refrigerated centrifugation (10000 rpm, 15 minutes), remove the upper clear liquid and disperse it in 1.0 mL of PBS. After refrigerated centrifugation (1000 rpm, 10 minutes), remove the lower substrate and add 400 uL of 3% BSA solution. Mix and react for 30 minutes to obtain specific CuO.

[0030] Step 6: Add the obtained 200uL specific magnetic beads and 400uL specific nano-CuO into 5mL of sheep leather protein solution with concentrations of 0.01g / mL, 0.001g / mL, and 0.0001g / mL, respectively, incubate with shaking at 37°C for 2h, remove the supernatant after centrifugation, place it in hydrogen peroxide, and pass the obtained oxygen into the pressure sensor.

[0031] The test results are as follows: the resistance of 0.01g / mL sheep leather protein solution is 30.25kΩ, the resistance of 0.001g / mL sheep leather protein solution is 37.76kΩ, and the resistance of 0.0001g / mL sheep leather protein solution is 46.87kΩ. The above data show that the specific magnetic beads and specific nano-CuO obtained in Example 1 can detect sheep leather protein in 0.01g / mL, 0.001g / mL, and 0.0001g / mL sheep leather protein solutions.

[0032] Comparative Example 1 (the only difference from Example 1 is that non-functionalized immunomagnetic beads are used)

[0033] Step 1: Mix 30 mL of 5 mg / mL copper nitrate solution and 30 mL of 10 mg / mL sodium carbonate solution, and disperse them by ultrasonication for 0.5 h. After reacting at 160 ° C for 4 h, vacuum filter the resulting powder, wash it with water and anhydrous ethanol, and finally dry it at 80 ° C for 1.5 h to obtain nano CuO.

[0034] Step 2: Add 2 mmol of ferric oxalate, 10 mol of dodecanediol, 0.8 g of dextran and 6 mol of oleic acid into 25 mL of ethanol to dissolve and obtain a mixed solution; react the obtained mixed solution at 220° C. for 20 min and then cool it to obtain a cooling solution containing a reaction product; wash the reaction product in the cooling solution to obtain immunomagnetic beads.

[0035] Step 3: Take 500 mg of immunomagnetic beads, add them to 100 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4, and disperse them by ultrasonic dispersion; add 200 mL of 23 wt% glutaraldehyde solution, and react in a water bath at 37°C for 2.5 h; magnetically separate the solid and liquid phases, wash them 5 times with PBS at pH 7.4, and disperse the activated magnetic beads in 50 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 to obtain a 10 mg / mL activated magnetic bead solution. Take 400 uL of the activated magnetic bead solution, add 100 uL of sheep leather protein antibody solution and shake it. After washing, add 200 uL of 3% BSA solution to mix and react. After washing, obtain specific magnetic beads, which are dispersed in 200 uL of phosphate buffer.

[0036] Step 4: Take 1.0 mg of nano-CuO and ultrasonicate it in 1.0 mL of PBS for 1 hour. Add 100 uL of sheep leather protein antibody to the obtained dispersion and shake it at 600 rpm for 4 hours. Centrifuge it at 10,000 rpm for 15 minutes and remove the upper clear liquid. Disperse it in 1.0 mL of PBS and centrifuge it at 1,000 rpm for 10 minutes. Remove the lower substrate and add 400 uL of 3% BSA solution. Mix and react for 30 minutes to obtain specific CuO.

[0037] Step 5: Add the obtained 200uL specific magnetic beads and 400uL specific nano-CuO into 5mL of sheep leather protein solution with concentrations of 0.01g / mL, 0.001g / mL, and 0.0001g / mL, respectively, incubate with shaking at 37°C for 2h, remove the supernatant after centrifugation, place it in hydrogen peroxide, and pass the obtained oxygen into the pressure sensor.

[0038] The test results are as follows: the resistance of 0.01g / mL sheep leather protein solution is 59.26kΩ, the resistance of 0.001g / mL sheep leather protein solution is 65.72kΩ, and the resistance of 0.0001g / mL sheep leather protein is 72.61kΩ of the sensor, which is close to the original resistance of the sensor 72.63kΩ. Therefore, it is shown that the detection limit of Comparative Example 1 is higher than 0.0001g / mL, revealing that the detection limit of the non-functional magnetic beads is higher than that of Example 1 using functionalized magnetic beads.

[0039] Example 2

[0040] A method for immunomagnetic enhancement detection of sheep leather comprises the following steps:

[0041] Step 1: Mix 30 mL of 5 mg / mL copper nitrate solution and 30 mL of 10 mg / mL sodium carbonate solution, and disperse them by ultrasonication for 0.5 h. After reacting at 160 ° C for 4 h, vacuum filter the resulting powder, wash it with water and anhydrous ethanol, and finally dry it at 80 ° C for 1.5 h to obtain nano CuO.

[0042] Step 2: Add 2 mmol of ferric oxalate, 10 mol of dodecanediol, 0.8 g of dextran and 6 mol of oleic acid into 25 mL of ethanol to dissolve and obtain a mixed solution; react the obtained mixed solution at 220° C. for 20 min and then cool it to obtain a cooling solution containing a reaction product; wash the reaction product in the cooling solution to obtain immunomagnetic beads.

[0043] Step 3: Take 0.4g of nano-immunomagnetic beads, add 20mL of water, and disperse evenly by ultrasonication. Then add 10mL of chloroform, 2.5mL of anhydrous ethanol, and then add 0.5g of acrylamide, 1.0mL of acetonitrile and 2.5mL of methacrylic acid. Stir rapidly for 20min at room temperature. After fully mixing, add 0.1g of initiator azobisisobutyronitrile, and stir rapidly at 45°C for 7h. Use an external magnetic field for solid-liquid separation to obtain functionalized nano-magnetic beads, and wash repeatedly with anhydrous ethanol and water until excess organic matter is washed away.

[0044] Step 4: Take 500 mg of functionalized nano-magnetic beads, add them to 100 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4, and disperse them by ultrasonic dispersion; add 200 mL of 23 wt% glutaraldehyde solution, and react in a water bath at 37°C for 2.5 h; magnetically separate the solid and liquid phases, wash them 5 times with PBS at pH 7.4, and disperse the activated magnetic beads in 50 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 to obtain a 10 mg / mL activated magnetic bead solution. Take 400 uL of the activated magnetic bead solution, add 100 uL of sheep leather protein antibody solution and shake it, add 200 uL of 3% BSA solution after washing, mix and react, and obtain specific magnetic beads after washing, which are dispersed in 200 uL of phosphate buffer.

[0045] Step 5: Take 1.0 mg of nano-CuO and ultrasonicate it in 1.0 mL of PBS for 1 hour. Add 100 uL of sheep leather protein antibody to the obtained dispersion and shake it at 600 rpm for 4 hours. After refrigerated centrifugation (10000 rpm, 15 minutes), remove the upper clear liquid and disperse it in 1.0 mL of PBS. After refrigerated centrifugation (1000 rpm, 10 minutes), remove the lower substrate and add 400 uL of 3% BSA solution. Mix and react for 30 minutes to obtain specific CuO.

[0046] Step 6: Add the obtained 200uL specific magnetic beads and 400uL specific nano-CuO into 5mL of sheep leather protein solution with concentrations of 100ng / mL, 10ng / mL, 1ng / mL, 0.1ng / mL, 0.01ng / mL, respectively, incubate at 37°C for 2h with shaking, remove the supernatant after centrifugation, place in hydrogen peroxide, and pass the obtained oxygen into the pressure sensor.

[0047] The test results are as follows: the resistance of 100ng / mL sheep leather protein solution is 49.63kΩ, the resistance of 10ng / mL sheep leather protein solution is 54.75kΩ, the resistance of 1ng / mL sheep leather protein solution is 59.47kΩ, the resistance of 0.1ng / mL sheep leather protein solution is 65.28kΩ, and the resistance of 0.01ng / mL sheep leather protein solution is 69.13kΩ. The above data show that the specific magnetic beads and specific nano-CuO obtained in Example 1 can detect sheep leather protein in the sheep leather protein solutions of the above five concentration gradients.

[0048] Example 3

[0049] A method for immunomagnetic enhancement detection of sheep leather comprises the following steps:

[0050] Step 1: Mix 30 mL of 5 mg / mL copper nitrate solution and 30 mL of 10 mg / mL sodium carbonate solution, and disperse them by ultrasonication for 0.5 h. After reacting at 160 ° C for 4 h, vacuum filter the resulting powder, wash it with water and anhydrous ethanol, and finally dry it at 80 ° C for 1.5 h to obtain nano CuO.

[0051] Step 2: Add 2 mmol of ferric oxalate, 10 mol of dodecanediol, 0.8 g of dextran and 6 mol of oleic acid into 25 mL of ethanol to dissolve and obtain a mixed solution; react the obtained mixed solution at 220° C. for 20 min and then cool it to obtain a cooling solution containing a reaction product; wash the reaction product in the cooling solution to obtain immunomagnetic beads.

[0052] Step 3: Take 0.4g of nano-immunomagnetic beads, add 20mL of water, and disperse evenly by ultrasonication. Then add 10mL of chloroform, 2.5mL of anhydrous ethanol, and then add 0.5g of acrylamide, 1.0mL of acetonitrile and 2.5mL of methacrylic acid. Stir rapidly for 20min at room temperature. After fully mixing, add 0.1g of initiator azobisisobutyronitrile, and stir rapidly at 45°C for 7h. Use an external magnetic field for solid-liquid separation to obtain functionalized nano-magnetic beads, and wash repeatedly with anhydrous ethanol and water until excess organic matter is washed away.

[0053] Step 4: Take 500 mg of functionalized nano-magnetic beads, add them to 100 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4, and disperse them by ultrasonic dispersion; add 200 mL of 23 wt% glutaraldehyde solution, and react in a water bath at 37°C for 2.5 h; magnetically separate the solid and liquid phases, wash them 5 times with PBS at pH 7.4, and disperse the activated magnetic beads in 50 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 to obtain a 10 mg / mL activated magnetic bead solution. Take 400 uL of the activated magnetic bead solution, add 100 uL of sheep leather protein antibody solution and shake it, add 200 uL of 3% BSA solution after washing, mix and react, and obtain specific magnetic beads after washing, which are dispersed in 200 uL of phosphate buffer.

[0054] Step 5: Take 1.0 mg of nano-CuO and ultrasonicate it in 1.0 mL of PBS for 1 hour. Add 100 uL of sheep leather protein antibody to the obtained dispersion and shake it at 600 rpm for 4 hours. After refrigerated centrifugation (10000 rpm, 15 minutes), remove the upper clear liquid and disperse it in 1.0 mL of PBS. After refrigerated centrifugation (1000 rpm, 10 minutes), remove the lower substrate and add 400 uL of 3% BSA solution. Mix and react for 30 minutes to obtain specific CuO.

[0055] Step 6: Add 200uL of the obtained specific magnetic beads and 400uL of specific nano-CuO to 5mLn 0.001g / mL silk fibroin solution, incubate with shaking at 37°C for 2h, remove the supernatant after centrifugation, place it in hydrogen peroxide, and pass the obtained oxygen into the pressure sensor.

[0056] The test results are as follows: the measured resistance is 72.63 kΩ, which is not significantly different from the original resistance of the sensor, while the resistance after adding 0.001 g / mL sheep leather protein is 37.45 kΩ, which indicates that this embodiment is specific to sheep leather protein and cannot detect silk protein.

[0057] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0058] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for immunomagnetic enhanced detection of sheep leather, characterized in that The following steps are involved: Step 1: Preparation of nano CuO: Mix copper nitrate solution and sodium carbonate solution, disperse by ultrasonication, react at 160-200°C for 4-8h, and then vacuum filter. Wash the obtained powder with water and anhydrous ethanol, and finally dry to obtain nano CuO. Step 2: Preparation of nano-immunomagnetic beads: adding ferric oxalate, dodecanediol, dextran and oleic acid to ethanol to dissolve to obtain a mixed solution; heating the obtained mixed solution for reaction and then cooling to obtain a cooling solution containing a reaction product; washing the reaction product in the cooling solution to obtain nano-immunomagnetic beads; Step 3: Preparation of functionalized nanomagnetic microparticles: Take nanoimmunomagnetic microparticles, add water, and disperse them evenly by ultrasonication. Then add chloroform, anhydrous ethanol, and then add acrylamide, acetonitrile and methacrylic acid. Stir rapidly at room temperature. After fully mixing, add initiator azobisisobutyronitrile, heat and stir rapidly to react, use an external magnetic field for solid-liquid separation, and obtain functionalized nanomagnetic microparticles. Wash repeatedly with anhydrous ethanol and water until excess organic matter is washed away. Step 4: Preparation of specific magnetic beads: adding functionalized nano magnetic beads into disodium hydrogen phosphate-sodium dihydrogen phosphate buffer and dispersing by ultrasonication; Add glutaraldehyde solution and shake in a water bath for reaction; The solid and liquid phases are magnetically separated, and washed with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, the activated magnetic beads are dispersed in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to obtain an activated magnetic bead solution, sheep leather protein antibody is added to the activated magnetic bead solution and shaken, BSA is added after washing and mixed to react, and specific magnetic beads are obtained after washing; Step 5: Preparation of specific CuO: Take nano-CuO and ultrasonicate it in PBS, add sheep leather protein antibody to the obtained dispersion, shake it; remove the upper clear liquid after freezing centrifugation, disperse it in PBS, remove the lower substrate after freezing centrifugation, add BSA solution, mix and react, and obtain specific CuO; Step 6: Add the obtained specific magnetic beads and specific CuO to the sheep leather protein solution to be tested, incubate with shaking, remove the supernatant after centrifugation, place in hydrogen peroxide, and pass the obtained oxygen into the pressure sensor.

2. The method according to claim 1, characterized in that: Step 1 is specifically as follows: 25-35 mL of 3-7 mg / mL copper nitrate solution and 25-35 mL of 8-12 mg / mL sodium carbonate solution are mixed and ultrasonically dispersed for 0.5-1 h, reacted at 160-200° C. for 4-8 h, vacuum filtered, and the obtained powder is washed with water and anhydrous ethanol successively, and finally dried at 80-100° C. for 1.5-2 h to obtain nano CuO.

3. The method according to claim 1, characterized in that: Step 2 is specifically as follows: 1.5-2.5 mmol of ferric oxalate, 8-12 mol of dodecanediol, 0.6-1.0 g of dextran and 4-8 mol of oleic acid are added to 20-30 mL of ethanol to dissolve to obtain a mixed solution; the obtained mixed solution is reacted at 180-250° C. for 5-30 min and then cooled to obtain a cooling solution containing a reaction product; The reaction product in the cooling solution is washed to obtain the immunomagnetic beads.

4. The method according to claim 1, characterized in that: Step 3 is specifically as follows: take 0.3-0.5g of nano-immunomagnetic beads, add 20mL of water, disperse evenly by ultrasonication, then add 8-12mL of chloroform, 2-3mL of anhydrous ethanol, then add 0.3-0.7g of acrylamide, 0.8-1.2mL of acetonitrile and 2-3mL of methacrylic acid, stir rapidly for 10-30min at room temperature, after fully mixing, add 0.08-0.12g of initiator azobisisobutyronitrile, stir rapidly at 40-50°C for 6-8h, perform solid-liquid separation using an external magnetic field to obtain functionalized nano-magnetic beads, and wash repeatedly with anhydrous ethanol and water until excess organic matter is washed away.

5. The method according to claim 1, characterized in that: Step 4 is specifically as follows: 400-600 mg of functionalized nano magnetic beads are taken, added to 80-120 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and ultrasonically dispersed; 150-250 mL of 20-30 wt% glutaraldehyde solution is added, and the reaction is oscillated in a water bath at 30-40° C. for 2-3 hours; the solid and liquid phases are magnetically separated, and the activated magnetic beads are washed 3-5 times with PBS, and the activated magnetic beads are dispersed in 40-60 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to obtain 8-12 mg / mL of activated magnetic bead solution, 350-450 uL of activated magnetic bead solution is separated, 80-120 uL of sheep leather protein antibody solution is added and shaken, and after washing, 150-250 uL of 2-4% BSA solution is added to mix and react, and specific magnetic beads are obtained after washing.

6. The method according to claim 1, characterized in that: Step 5 is specifically as follows: 0.8-1.2 mg of nano-CuO is taken in 0.8-1.2 mL of PBS and ultrasonicated for 0.5-1.5 h, 80-120 uL of sheep leather protein antibody is added to the obtained dispersion, and the dispersion is shaken at 500-700 rpm for 3-5 h; the supernatant is removed after freeze centrifugation, and the dispersion is dispersed in 0.8-1.2 mL of PBS, and the lower substrate is removed after freeze centrifugation, and 350-450 uL of 2-4% BSA solution is added, and the mixture is mixed and reacted for 20-40 min to obtain specific CuO.

7. The method according to claim 6, characterized in that: In step 5, the first centrifugation rate is 8000-12000 rpm, and the time is 10-20 min; the second centrifugation rate is 800-1200 rpm, and the time is 5-15 min.

8. The method according to claim 1, characterized in that: Step 6 is specifically as follows: adding the obtained specific magnetic beads and specific nano-CuO to the sheep leather protein solution to be detected, incubating with shaking at 30-40°C for 1-3h, removing the supernatant after centrifugation, placing in hydrogen peroxide, and passing the obtained oxygen into the pressure sensor.