Method for detecting leather textile cultural relics based on flexible pressure immune sensor

Abstract

The present application relates to the technical field of leather textile cultural relic detection, and discloses a method for detecting leather textile cultural relics based on a flexible pressure immune sensor, comprising the following steps: step 1, preparation of a nano copper oxide antibody probe; step 2, preparation of a carboxylated magnetic bead immune probe; step 3, preparation of a three-dimensional polypyrrole foam; step 4, preparation of a polypyrrole foam flexible pressure sensor; and step 5, detection of leather collagen: dissolving leather collagen powder with a phosphate buffer solution, adding a nano copper oxide probe dispersion and a carboxylated magnetic bead immune probe dispersion, and incubating at room temperature; after incubation, adding H2O2 for reaction, obtaining an electric response of pressure sensing from a digital multimeter, and then obtaining the concentration of leather collagen. Through specific binding of antigens and antibodies, the present application can exclude the interference of various impurities, reduce the detection limit, and improve detection specificity and accuracy.

Description

Technical Field

[0001] This invention belongs to the technical field of testing leather textile artifacts, and more specifically relates to a method for testing leather textile artifacts based on a flexible pressure immunosensor. Background Technology

[0002] my country is one of the earliest countries to use textiles. These textiles bear witness to the social changes, economic development, and cultural integration of my country, and are invaluable historical materials for studying the economic, technological, and cultural level of ancient my country. Most of the textile artifacts discovered so far are protein-based, with leather textiles being particularly susceptible to aging and decomposition due to the influence of moisture, temperature, pH levels, and microorganisms in the burial environment, resulting in severe damage. Therefore, finding a highly sensitive, specific, simple, quick, and efficient method for identifying the species of leather artifacts is of great significance for studying the origins of leather artifacts in my country, and also provides new scientific evidence for the study of thousands of years of Chinese clothing culture. Summary of the Invention

[0003] In view of this, to achieve the above objectives, the present invention provides a method for detecting leather textile artifacts based on a flexible pressure immunosensor, comprising the following steps:

[0004] Step 1: Preparation of nano-copper oxide antibody probe: Dissolve nano-copper oxide in phosphate buffer solution, disperse by sonication, add leather collagen antibody, shake and freeze-centrifuge, remove supernatant and disperse in phosphate buffer solution, freeze-centrifuge again, take supernatant and add antibody blocking solution and shake to obtain nano-copper oxide probe dispersion, store in cold storage.

[0005] Step 2, Preparation of Carboxylated Magnetic Bead Immunoplasmic Probes: The carboxylated magnetic bead solution was washed with phosphate wash buffer, and then redispersed in phosphate buffer to obtain a carboxylated magnetic bead dispersion. 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added to MES buffer, and the mixture was added to the carboxylated magnetic bead dispersion for isothermal incubation. After incubation, the activated magnetic beads were washed with phosphate wash buffer, dispersed in phosphate buffer, and then coated with leather collagen-coated antibody for another isothermal incubation. After incubation, antibody blocking solution was added, and the beads were shaken to block excess sites on the magnetic beads. After magnetic separation and washing with phosphate wash buffer, the beads were redispersed in phosphate buffer to obtain a carboxylated magnetic bead immunoprobe dispersion, which was then refrigerated.

[0006] Step 3: Preparation of three-dimensional polypyrrole foam: Pyrrole was added to NaClO4 solution and ultrasonically treated to obtain a pyrrole solution; using nickel foam as the working electrode, and Pt and Ag / AgCl as the counter and reference electrodes respectively, the pyrrole solution was added to perform an electrodeposition reaction to obtain PPy / Ni foam; after washing and drying, the PPy / Ni foam was immersed in PDMS solution, then removed and excess PDMS solution was removed before curing; the cured PPy / Ni foam was immersed in a solution containing FeCl3 and HCl, degassed, and allowed to stand; then it was removed, washed, and dried to obtain three-dimensional polypyrrole foam;

[0007] Step 4: Fabrication of the polypyrrole foam flexible pressure sensor: Three-dimensional polypyrrole foam slices are cut, and conductive ITO / PET films are covered on the top and bottom with silver paste. Two copper strip electrodes are connected to the ITO / PET films as electrical contacts. The whole thing is then wrapped with Capton tape and PDMS, placed in a container, and connected to a digital multimeter to obtain the polypyrrole foam flexible pressure sensor.

[0008] Step 5: Detection of leather collagen: Dissolve leather collagen powder in phosphate buffer solution, add nano copper oxide probe dispersion and carboxylated magnetic bead immunoprobe dispersion, and incubate at room temperature; after incubation, adsorb it using a magnetic rack, wash repeatedly with phosphate buffer, and then react in H2O2. Obtain the electrical response of the pressure sensor from a digital multimeter, and then obtain the concentration of leather collagen.

[0009] In step 2, this invention adds EDC and NHS as amino and carboxyl coupling agents to activate the carboxyl groups on the magnetic beads, enabling them to bind to the amino groups on the antibody surface. This further enhances the ability of the magnetic beads to bind proteins, lowers the detection limit, and allows detection even at trace protein concentrations. In step 3, this invention adds PDMS as a curing agent, giving the three-dimensional polypyrrole foam good mechanical strength, further improving its durability and reproducibility. Finally, utilizing the principle of CuO catalysis of hydrogen peroxide, the oxygen generated by the catalytic action of nano-copper oxide on hydrogen peroxide is introduced into the flexible pressure sensor, causing a pressure change within the container. The electrical response of the pressure sensor is obtained from a digital multimeter, significantly improving detection efficiency.

[0010] Further preferred, step 1 specifically involves: dissolving 1-5 mg of nano-copper oxide in 2-3 mL of pH 7.4 phosphate buffer solution, ultrasonically dispersing for 4-7 h, then adding 100-400 μL of rabbit anti-leather collagen antibody solution with a concentration of 2.0 mg / mL, shaking for 5-7 h, followed by freezing and centrifugation. After removing the supernatant, disperse the solution in 2-3 mL of pH 7.4 phosphate buffer solution, and freeze and centrifuge again. After centrifugation, take the supernatant, add 100-400 μL of antibody blocking solution, and shake to obtain the nano-copper oxide probe dispersion, which is then stored at 4°C.

[0011] Further preferably, step 2 specifically involves: washing 10-30 μL of a 40-70 mg / mL carboxylated magnetic bead solution with phosphate wash buffer, and then redispersing it in 300 μL of pH 7.4 phosphate buffer to obtain a carboxylated magnetic bead dispersion; adding 13-16 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-7 mg of N-hydroxysuccinimide to 5-20 mL of pH 6-8 MES buffer, and adding 300 μL of this mixture to the carboxylated magnetic bead dispersion, and incubating at 37°C for 40-50 min; The activated magnetic beads were then washed with phosphate wash buffer and dispersed in 300 μL of pH 7.4 phosphate buffer. 20-50 μL of mouse anti-leather collagen-coated antibody solution (0.6-1.7 nmol / mL) was added, and the mixture was incubated again at 37°C for 90-100 min. After incubation, 100-400 μL of antibody blocking solution was added, and the mixture was shaken for 1-1.5 h to block excess sites on the magnetic beads. After magnetic separation and washing with phosphate wash buffer, the mixture was redispersed in 500 μL of pH 7.4 phosphate buffer to obtain a carboxylated magnetic bead immunoprobe dispersion, which was stored at 4°C.

[0012] Further preferably, step 3 specifically involves: adding 1.7-4.6 mL of pyrrole to 30-60 mL of a 0.24-0.42 M NaClO4 solution and sonicating to obtain a pyrrole solution; using a 0.7-2.3 mm thick nickel foam as the working electrode, and Pt and Ag / AgCl as the counter and reference electrodes, respectively, adding the pyrrole solution and electrodepositing it at a potential of 0.4-0.9 V for 300-700 s to obtain PPy / Ni foam; washing and drying the PPy / Ni foam, immersing it in a PDMS solution for 30-40 min, then removing it and removing excess PDMS solution, and curing it at 70-80 °C for 2-3 h; immersing the cured PPy / Ni foam in a solution containing FeCl3 and HCl, degassing it for 3-5 min, and then letting it stand for 24-30 h; then removing it, washing it, and drying it to obtain three-dimensional polypyrrole foam;

[0013] More preferably, the PDMS solution is a diluted solution of hexane and polydimethylsiloxane mixed in a weight ratio of 40-60:1-8.

[0014] More preferably, in the solution containing FeCl3 and HCl, the concentration of FeCl3 is 1-4M and the concentration of HCl is 1-3M.

[0015] In a further preferred embodiment, step 5 specifically involves: dissolving leather collagen powder in 1-5 mL of phosphate buffer solution, adding 100-400 μL of nano-copper oxide probe dispersion and 200-500 μL of carboxylated magnetic bead immunoprobe dispersion, and incubating at room temperature for 10-50 min; after incubation, adsorbing the material using a magnetic rack, repeatedly washing with phosphate buffer solution, and then reacting in 100-300 μL of H2O2 for 6-8 min, obtaining the electrical response of the pressure sensor from a digital multimeter, and thus obtaining the concentration of leather collagen.

[0016] Compared with existing technologies, the present invention has the following advantages: 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; the present invention utilizes the principle of CuO catalyzing hydrogen peroxide, which can significantly improve the detection efficiency; by utilizing the paramagnetism of magnetic beads, the synthesized materials and reaction system can be better separated, which facilitates the washing and purification of the synthesized materials. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the present invention's scheme for detecting leather textile artifacts based on a flexible pressure immunosensor;

[0018] Figure 2 This is a calibration graph showing the change in relative resistance as a function of the standard concentration index of leather collagen in Example 1;

[0019] Figure 3 This is for the specific detection of different types of proteins in Example 2 (10 μg / mL for each case);

[0020] Figure 4 The results of detecting collagen in leather using different conditions and amounts are shown in Comparative Example 1.

[0021] Figure 5 The results of detecting collagen in leather after using untreated carboxylated magnetic beads in Comparative Example 2 are shown.

[0022] Figure 6 The results of detecting collagen in leather after using untreated copper oxide in Comparative Example 3;

[0023] Figure 7 The results of detecting collagen in leather in Comparative Example 4 without the use of H2O2 are shown. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the embodiments, and the technical content and effects thereof are not limited thereto.

[0025] Example 1

[0026] A method for detecting leather textile artifacts based on a flexible pressure immunosensor includes the following steps:

[0027] Step 1: Dissolve 2 mg of nano-copper oxide in 1.5 mL of phosphate buffer (pH = 7.4), then sonicate in an ultrasonic cell disruptor for 6 h. After sonication, add 200 μL of 2.0 mg / mL leather collagen antibody solution and shake on a shaker for 5 h. Then place the nano-copper oxide solution containing leather collagen antibody in a high-speed refrigerated centrifuge and centrifuge at 12000 rpm for 20 min. Remove the supernatant from the centrifuged mixture to remove any leather collagen antibody that is not conjugated with nano-copper oxide. The remaining precipitate was then redispersed in 1.5 mL of phosphate buffer (pH = 7.4). The dispersed solution was centrifuged at 1000 rpm for 8 min. After centrifugation, the supernatant was collected to remove excess copper oxide nanoparticles. 300 μL of antibody blocking solution (PBS solution with BSA concentration of 1 wt%) was added to the supernatant, and the mixture was shaken on a shaker for 1 h to obtain the copper oxide nanoparticle antibody probe dispersion, which was then stored in a refrigerator at 4 °C.

[0028] Step 2: Transfer 10 μL of a 50 mg / mL carboxylated magnetic bead solution to a 1.5 mL microcentrifuge tube using a pipette. Then, use a pipette to wash the magnetic beads with 300 μL of phosphate wash buffer containing 0.05 wt% Tween 20 three times. After washing, redisperse the magnetic beads in 300 μL of phosphate buffer (pH = 7.4) to obtain a carboxylated magnetic bead dispersion. Next, add 14.59 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 5.41 mg of N-hydroxysuccinimide (NHS) to 10 mL of LME buffer (pH = 6.0). Then, transfer 300 μL of this mixture to the carboxylated magnetic bead dispersion and incubate the reaction at 37 °C for 40 min. After activation, the magnetic beads were washed three times with phosphate wash buffer containing 0.05 wt% Tween 20, then dispersed in 300 μL of phosphate buffer (pH = 7.4). Next, 40 μL of mouse anti-leather collagen-coated antibody (1.0 mg / mL) was added, and the mixture was incubated at 37°C for 90 min. Then, 100 μL of antibody blocking solution (1 wt% BSA in PBS solution) was added, and the mixture was shaken for 1 h to block excess active sites on the magnetic beads. Finally, the antibody-functionalized magnetic beads were magnetically separated and washed three times with phosphate wash buffer containing 0.05% Tween 20, then redispersed in 500 μL of phosphate buffer (pH = 7.4) to obtain a carboxylated magnetic bead immunoprobe dispersion, which was stored at 4°C.

[0029] Step 3: Add 2.5 mL of pyrrole (note that pyrrole should be distilled before use) to 50 mL of NaClO4 (0.3 M) solution and sonicate for 10 min to form a pale yellow pyrrole solution. Use nickel foam (1.5 mm thick) as the working electrode, and Pt and Ag / AgCl as the counter and reference electrodes, respectively. Add them to the pyrrole solution and electrodeposit at a potential of 0.8 V for 400 s. After deposition, rinse the obtained PPy / Ni foam with ethanol and water, dry it after hysteresis, and then immerse it in a diluted PDMS solution prepared by mixing hexane and polydimethylsiloxane (containing curing agent) at a weight ratio of 60:1 for 30 min. After removing excess PDMS solution, cure it at 70 °C for 3 h. Immerse the PDMS-coated PPy / Ni foam in an aqueous solution containing FeCl3 (3 M) and HCl (1 M) for 3 min to degas it, allowing the solution to enter the gaps. After standing for 24 h, take it out, rinse it with ethanol, and dry it at 60 °C to obtain three-dimensional polypyrrole foam.

[0030] Step 4: Cut the three-dimensional polypyrrole foam into pieces (1.2 × 1.2 cm). 2The polypyrrole foam flexible pressure sensor is fabricated by covering its top and bottom with two indium tin oxide / polyethylene terephthalate (ITO / PET) films using silver paste, and attaching two copper strip electrodes to the ITO / PET films as electrical contacts. The entire assembly is then wrapped with Capton tape and PDMS. A container is printed using a 3D printer, and the sensor is placed inside the printed container and connected to a digital multimeter (DMM) to complete the fabrication.

[0031] Step 5: Take 2 mL of leather collagen solutions with concentrations of 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL, respectively, and add 300 μL of the nano-copper oxide probe dispersion prepared in Step 1 and 500 μL of the carboxylated magnetic bead immunoprobe dispersion prepared in Step 2. Incubate at room temperature for 40 min, and after adsorption using a magnetic rack, wash three times with 300 μL of phosphate wash buffer. Place the washed product in a polypyrrole foam flexible pressure sensor, add 200 μL of H2O2, react for 6 min, and obtain the electrical response of the pressure sensor from the DMM to determine the concentration of leather collagen.

[0032] Example 2

[0033] A method for detecting leather textile artifacts based on a flexible pressure immunosensor includes the following steps:

[0034] Steps 1-4 are exactly the same as in Example 1.

[0035] Step 5: Take 2 mL each of 10 μg / mL leather solution, silk solution, cotton solution, and hemp solution, and 2 mL of PBS. Add 300 μL of the nano-copper oxide probe dispersion prepared in Step 1 and 500 μL of the carboxylated magnetic bead immunoprobe dispersion prepared in Step 2 to each solution. Incubate at room temperature for 40 min. After adsorption using a magnetic rack, wash three times with 300 μL of phosphate wash buffer. Place the washed product in a polypyrrole foam flexible pressure sensor, add 200 μL of H2O2, and react for 6 min. Obtain the electrical response of the pressure sensor from the DMM to obtain the collagen concentration.

[0036] Example 3

[0037] A method for detecting leather textile artifacts based on a flexible pressure immunosensor includes the following steps:

[0038] Step 1: Dissolve 1 mg of nano-copper oxide in 1.5 mL of phosphate buffer (pH = 7.4), then sonicate in an ultrasonic cell disruptor for 6 h. After sonication, add 300 μL of 2.0 mg / mL leather collagen antibody solution and shake on a shaker for 5 h. Then place the nano-copper oxide solution containing leather collagen antibody in a high-speed refrigerated centrifuge and centrifuge at 12000 rpm for 20 min. Remove the supernatant from the centrifuged mixture to remove leather collagen antibody that is not coupled with nano-copper oxide. The remaining precipitate was then redispersed in 1.5 mL of phosphate buffer (pH = 7.4). The dispersed solution was centrifuged at 1000 rpm for 8 min. After centrifugation, the supernatant was collected to remove excess copper oxide nanoparticles. 400 μL of antibody blocking solution (PBS solution with BSA concentration of 1 wt%) was added to the supernatant, and the mixture was shaken for 1 h to obtain the copper oxide nanoparticle antibody probe dispersion, which was then stored in a refrigerator at 4 °C.

[0039] Step 2: Transfer 10 μL of a 100 mg / mL carboxylated magnetic bead solution to a 1.5 mL microcentrifuge tube using a pipette. Then, use a pipette to wash the magnetic beads with 300 μL of phosphate wash buffer containing 0.05 wt% Tween 20 three times. After washing, redisperse the magnetic beads in 300 μL of phosphate buffer (pH = 7.4) to obtain a carboxylated magnetic bead dispersion. Next, add 14.59 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 5.41 mg of N-hydroxysuccinimide (NHS) to 5 mL of LME buffer (pH = 6.0). Then, transfer 300 μL of this mixture to the carboxylated magnetic bead dispersion and incubate the reaction at 37 °C for 40 min. After activation, the magnetic beads were washed three times with phosphate wash buffer containing 0.05 wt% Tween 20, then dispersed in 300 μL of phosphate buffer (pH = 7.4). Next, 40 μL of mouse anti-leather collagen-coated antibody (4.0 mg / mL) was added, and the mixture was incubated at 37°C for 90 min. Then, 300 μL of antibody blocking solution (1 wt% BSA in PBS solution) was added, and the mixture was shaken for 1 h to block excess active sites on the magnetic beads. Finally, the antibody-functionalized magnetic beads were magnetically separated and washed three times with phosphate wash buffer containing 0.05% Tween 20, then redispersed in 500 μL of phosphate buffer (pH = 7.4) to obtain a carboxylated magnetic bead immunoprobe dispersion, which was stored at 4°C.

[0040] Step 3: Add 2.5 mL of pyrrole (note that pyrrole should be distilled before use) to 50 mL of NaClO4 (0.3 M) solution and sonicate for 10 min to form a pale yellow pyrrole solution. Use nickel foam (1.5 mm thick) as the working electrode, and Pt and Ag / AgCl as the counter and reference electrodes, respectively. Add them to the pyrrole solution and electrodeposit them at a potential of 0.5 for 300 s. After deposition, rinse the obtained PPy / Ni foam with ethanol and water, dry it after hysteresis, and then immerse it in a diluted PDMS solution prepared by mixing hexane and polydimethylsiloxane (containing curing agent) at a weight ratio of 60:1 for 30 min. After removing excess PDMS solution, cure it at 70 °C for 3 h. With the help of a magnet, immerse the PDMS-coated PPy / Ni foam in an aqueous solution containing FeCl3 (3 M) and HCl (1 M) for 3 min to degas, allowing the solution to enter the gaps. After standing for 24 hours, the product was taken out, rinsed with ethanol, and dried at 60°C to obtain three-dimensional polypyrrole foam.

[0041] Step 4: Cut the three-dimensional polypyrrole foam into (1.2×1.2cm²) sheets. Cover the top and bottom of each sheet with two indium tin oxide / polyethylene terephthalate (ITO / PET) films using silver paste. Connect two copper strip electrodes to the ITO / PET films as electrical contacts. Wrap the entire sheet with Capton tape and PDMS. Print a container using a 3D printer, place the polypyrrole foam sheet inside the printed container, and connect it to a digital multimeter (DMM) to complete the fabrication of the polypyrrole foam flexible pressure sensor.

[0042] Step 5: Take 2 mL of leather collagen solutions with concentrations of 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL, respectively. Add 300 μL of the nano-copper oxide probe dispersion prepared in Step 1 and 500 μL of the carboxylated magnetic bead immunoprobe dispersion prepared in Step 2 to each solution. Incubate at room temperature for 40 min. After adsorption using a magnetic rack, wash three times with 300 μL of phosphate wash buffer. Place the washed product in a polypyrrole foam flexible pressure sensor, add 200 μL of H2O2, react for 6 min, and obtain the electrical response of the pressure sensor from the DMM to determine the leather collagen concentration.

[0043] Comparative Example 1

[0044] A method for detecting leather textile artifacts based on a flexible pressure immunosensor includes the following steps:

[0045] Step 1: Dissolve 1 mg of nano-copper oxide in 1.5 mL of phosphate buffer (pH = 7.4), then sonicate in an ultrasonic cell disruptor for 6 h. After sonication, add 200 μL of 2.0 mg / mL leather collagen antibody solution and shake on a shaker for 5 h. Then place the nano-copper oxide solution containing leather collagen antibody in a high-speed refrigerated centrifuge and centrifuge at 12000 rpm for 20 min. Remove the supernatant from the centrifuged mixture to remove the leather collagen antibody that is not coupled with nano-copper oxide. The remaining precipitate was then redispersed in 1.5 mL of phosphate buffer (pH = 7.4). The dispersed solution was centrifuged at 1000 rpm for 8 min. After centrifugation, the supernatant was collected to remove excess copper oxide nanoparticles. 300 μL of antibody blocking solution (PBS solution with BSA concentration of 1 wt%) was added to the supernatant, and the mixture was shaken on a shaker for 1 h to obtain the copper oxide nanoparticle antibody probe dispersion, which was then stored in a refrigerator at 4 °C.

[0046] Step 2: Transfer 10 μL of a 100 mg / mL carboxylated magnetic bead solution to a 1.5 mL microcentrifuge tube using a pipette. Then, use a pipette to wash the magnetic beads with 300 μL of phosphate wash buffer containing 0.05 wt% Tween 20 three times. After washing, redisperse the magnetic beads in 300 μL of phosphate buffer (pH = 7.4) to obtain a carboxylated magnetic bead dispersion. Next, add 14.59 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 5.41 mg of N-hydroxysuccinimide (NHS) to 5 mL of LME buffer (pH = 6.0). Then, transfer 300 μL of this mixture to the carboxylated magnetic bead dispersion and incubate the reaction at 37 °C for 40 min. After activation, the magnetic beads were washed three times with phosphate wash buffer containing 0.05 wt% Tween 20, then dispersed in 300 μL of phosphate buffer (pH = 7.4). Next, 40 μL of mouse anti-leather collagen-coated antibody (4.0 mg / mL) was added, and the mixture was incubated at 37°C for 90 min. Then, 100 μL of antibody blocking solution (1 wt% BSA in PBS solution) was added, and the mixture was shaken for 1 h to block excess active sites on the magnetic beads. Finally, the antibody-functionalized magnetic beads were magnetically separated and washed three times with phosphate wash buffer containing 0.05% Tween 20, then redispersed in 500 μL of phosphate buffer (pH = 7.4) to obtain a carboxylated magnetic bead immunoprobe dispersion, which was stored at 4°C.

[0047] Steps 3-5 are exactly the same as steps 3-5 in Example 1.

[0048] Comparative Example 2

[0049] A method for detecting leather textile artifacts based on a flexible pressure immunosensor includes the following steps:

[0050] Steps 1-3 are exactly the same as steps 1, 3, and 4 in Example 1.

[0051] Step 4: Take 2 mL of leather collagen solutions with concentrations of 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL, respectively, and add 300 μL of the nano-copper oxide probe dispersion prepared in Step 1. Incubate at room temperature for 40 min, then adsorb using a magnetic rack and wash three times with 300 μL of phosphate wash buffer. Place the washed product in a polypyrrole foam flexible pressure sensor, add 200 μL of H2O2, react for 6 min, and obtain the electrical response of the pressure sensor from the DMM to determine the leather collagen concentration.

[0052] Comparative Example 3

[0053] A method for detecting leather textile artifacts based on a flexible pressure immunosensor includes the following steps:

[0054] Step 1: Dissolve 2 mg of nano-copper oxide in 1.5 mL of phosphate buffer (pH = 7.4). Then, place the mixture in an ultrasonic cell disruptor and sonicate for 6 hours. After sonication, shake on a shaker for 5 hours, then place in a high-speed refrigerated centrifuge and centrifuge at 12,000 rpm for 20 minutes. Remove the supernatant from the centrifuged mixture, then redisperse the precipitate in 1.5 mL of phosphate buffer (pH = 7.4). Centrifuge the dispersed solution at 1,000 rpm for 8 minutes, collect the supernatant, add 300 μL of antibody blocking solution to the supernatant, and continue shaking on a shaker for 1 hour to obtain the nano-copper oxide probe dispersion. Store in a refrigerator at 4°C.

[0055] Steps 2-4 are exactly the same as steps 2-4 in Example 1.

[0056] Step 5: Take 2 mL of leather collagen solutions with concentrations of 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL, respectively, and add 300 μL of the nano-copper oxide probe dispersion prepared in Step 1 and 500 μL of the carboxylated magnetic bead immunoprobe dispersion prepared in Step 2. Incubate at room temperature for 40 min, and after adsorption using a magnetic rack, wash three times with 300 μL of phosphate wash buffer. Place the washed product in a polypyrrole foam flexible pressure sensor, add 200 μL of H2O2, react for 6 min, and obtain the electrical response of the pressure sensor from the DMM to determine the concentration of leather collagen.

[0057] Comparative Example 4

[0058] A method for detecting leather textile artifacts based on a flexible pressure immunosensor includes the following steps:

[0059] Steps 1-4 are exactly the same as steps 1-4 in Example 1.

[0060] Step 5: Take 2 mL of leather collagen solutions with concentrations of 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL, respectively. Add 300 μL of the nano-copper oxide probe dispersion prepared in Step 1 and 500 μL of the carboxylated magnetic bead immunoprobe dispersion prepared in Step 2 to each solution. Incubate at room temperature for 40 min. After adsorption using a magnetic rack, wash three times with 300 μL of phosphate wash buffer. Place the washed product in a polypyrrole foam flexible pressure sensor, add 200 μL of H2O, react for 6 min, and obtain the electrical response of the pressure sensor from the DMM to determine the leather collagen concentration.

[0061] like Figure 1 The diagram shows a schematic representation of the present invention's method for detecting leather textile artifacts using a flexible pressure immunosensor. Figure 2 This indicates that the flexible pressure sensor obtained in Example 1 can be used to detect leather textile artifacts, with a detection limit of 13.2 ng / mL.

[0062] Depend on Figure 3 This indicates that the flexible pressure immunosensor obtained in Example 2 is specific for leather collagen. Under the same preparation and detection conditions, there is a significant change in resistance when detecting leather collagen, while there is no significant change when detecting other textile proteins, indicating that it can specifically recognize leather collagen.

[0063] Depend on Figure 4The results show that, after changing the conditions and amounts added in Comparative Example 1 (specifically: using 1 mg of nano-copper oxide and 200 μL of leather collagen antibody to prepare a nano-copper oxide antibody probe dispersion; using a carboxylated magnetic bead solution with a concentration of 100 mg / mL, 5 mL of MES solution containing EDC / NHS, and a mouse anti-leather collagen coated antibody with a concentration of 4.0 mg / mL to prepare a carboxylated magnetic bead immunoprobe dispersion), the detection effect of Comparative Example 1 for detecting leather collagen was significantly weaker than that of Example 1.

[0064] Depend on Figure 5 This indicates that, in Comparative Example 2, the final test without the addition of carboxylated magnetic bead immune probes failed to detect the presence of leather collagen.

[0065] Depend on Figure 6 This indicates that the addition of ordinary nano-copper oxide in the final test of Comparative Example 3 did not detect the presence of leather collagen.

[0066] Depend on Figure 7 This indicates that in Comparative Example 4, no hydrogen peroxide was added in the final test. With the addition of H2O, copper oxide ions could not catalyze the generation of oxygen from water, thus failing to generate pressure in the sensor's enclosed space and making it impossible to detect the presence of leather collagen.

[0067] The above embodiments of the present invention are merely illustrative examples and are not intended to limit the implementation of the invention. Those skilled in the art can make other variations and modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for detecting leather textile artifacts based on a flexible pressure immunosensor, characterized in that, Comprising the following steps: Step 1, preparation of nano copper oxide antibody probe: 1-5 mg of nano copper oxide is dissolved in 2-3 mL of pH=7.4 phosphate buffer solution, ultrasonic dispersion for 4-7 h, then 100-400 μL of 2.0 mg / mL rabbit anti-leather collagen antibody solution is added, and after oscillation for 5-7 h, it is frozen and centrifuged, the supernatant is removed and dispersed in 2-3 mL of pH=7.4 phosphate buffer solution, and then frozen and centrifuged again, the supernatant is taken after centrifugation, 100-400 μL of antibody blocking solution is added and oscillated, and the nano copper oxide probe dispersion is obtained, which is stored at 4°C; Step 2, preparation of carboxylated magnetic bead immune probe: 10-30 μL of 40-70 mg / mL carboxylated magnetic bead solution is washed with phosphate washing buffer, and after washing, it is dispersed in 300 μL of pH=7.4 phosphate buffer solution to obtain a carboxylated magnetic bead dispersion; 13-16 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 4-7 mg of N-hydroxysuccinimide are added to 5-20 mL of pH=6-8 MES buffer solution, 300 μL of the mixture is added to the carboxylated magnetic bead dispersion, and incubated at 37°C for 40-50 min; the activated magnetic beads are washed with phosphate washing buffer, dispersed in 300 μL of pH=7.4 phosphate buffer solution, and 20-50 μL of 0.6-1.7 mg / mL mouse anti-leather collagen coating antibody solution is added, and incubated at 37°C for 90-100 min; after incubation, 100-400 μL of antibody blocking solution is added and oscillated for 1-1.5 h to block the excess sites on the magnetic beads, which are separated by magnetism and washed with phosphate washing buffer, and then dispersed in 500 μL of pH=7.4 phosphate buffer solution to obtain a carboxylated magnetic bead immune probe dispersion, which is stored at 4°C; Step 3, preparation of three-dimensional polypyrrole foam: pyrrole is added to NaClO4 solution, ultrasonic is used to obtain pyrrole solution; foam nickel is used as the working electrode, Pt and Ag / AgCl are used as the counter electrode and reference electrode respectively, pyrrole solution is added, and electrodeposition reaction is carried out to obtain PPy / Ni foam; the PPy / Ni foam is washed, dried, immersed in PDMS solution, the PDMS solution is a dilute solution mixed by hexane and polydimethylsiloxane at a weight ratio of 40-60:1-8, then removed and solidified; the solidified PPy / Ni foam is immersed in a solution containing FeCl3 and HCl, the concentration of FeCl3 in the solution containing FeCl3 and HCl is 1-4 M, and the concentration of HCl is 1-3 M, and the solution is degassed and left standing; then it is taken out, washed and dried to obtain three-dimensional polypyrrole foam; Step 4, preparation of polypyrrole foam flexible pressure sensor: cut the three-dimensional polypyrrole foam into slices, cover the top and bottom of the slices with silver paste respectively, and cover ITO / PET film and two copper strip electrodes as electrical contacts; then wrap the whole with kapton tape and PDMS, put it into a container, connect a digital multimeter, and obtain a polypyrrole foam flexible pressure sensor; Step 5, detection of leather collagen: dissolve the leather collagen powder with a phosphate buffer solution, add a nano-copper oxide probe dispersion and a carboxylated magnetic bead immune probe dispersion, and incubate at room temperature; after incubation, use a magnetic stand to adsorb it, and then repeatedly wash it with a phosphate buffer solution; place it in a polypyrrole foam flexible pressure sensor, add H2O2 for reaction, obtain the electrical response of pressure sensing from a digital multimeter, and then obtain the concentration of leather collagen.

2. The method for detecting leather textile artifacts based on flexible pressure immunosensor according to claim 1, characterized in that, Step 3 specifically comprises: adding 1.7-4.6 mL of pyrrole to 30-60 mL of NaClO4 solution with a concentration of 0.24-0.42 M to obtain a pyrrole solution by ultrasonic; using a foam nickel with a thickness of 0.7-2.3 mm as a working electrode, Pt and Ag / AgCl as a counter electrode and a reference electrode respectively, adding the pyrrole solution, and performing electrodeposition at a potential of 0.4-0.9 V for 300-700 s to obtain a PPy / Ni foam; after washing and drying the PPy / Ni foam, immerse it in a PDMS solution for 30-40 min, then take it out and remove the excess PDMS solution, and solidify it at 70-80°C for 2-3 h; immerse the solidified PPy / Ni foam in a solution containing FeCl3 and HCl, deaerate for 3-5 min, and then stand for 24-30 h; then take it out, wash and dry it to obtain a three-dimensional polypyrrole foam.

3. The method for detecting leather textile artifacts based on flexible pressure immunosensor according to claim 1, characterized in that, Step 5 specifically comprises: dissolving the leather collagen powder with 1-5 mL of a phosphate buffer solution, adding 100-400 μL of a nano-copper oxide probe dispersion and 200-500 μL of a carboxylated magnetic bead immune probe dispersion, and incubating at room temperature for 10-50 min; after incubation, use a magnetic stand to adsorb it, and then repeatedly wash it with a phosphate buffer solution, add 100-300 μL of H2O2 for reaction for 6-8 min, obtain the electrical response of pressure sensing from a digital multimeter, and then obtain the concentration of leather collagen.

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