Preparation method of electrochemical immunosensor for detecting leather cultural relics

Abstract

The invention relates to the technical field of immunosensors, and discloses a preparation method of an electrochemical immunosensor for detecting leather cultural relics, and the preparation method comprises the following steps: step 1, preparing MXene / CNTs; step 2, preparing an MOFs (Metal-Organic Frameworks) material; step 3, adding glutaraldehyde stationary liquid into the obtained MOFs solution for reaction; adding a streptavidin solution into the mixed solution a and reacting; adding a biotin-labeled antibody solution into the obtained mixed solution b to react, so as to prepare MOFs-Ab2; step 4, dropwise adding the MXene / CNTs dispersion liquid on a glassy carbon electrode, drying, then placing the glassy carbon electrode in an Ab1 solution to absorb an antibody, and then placing the glassy carbon electrode in a BSA solution to close a site; then, a leather cultural relic sample solution is dropwise added, and finally, the leather cultural relic sample solution is placed in MOFs-Ab2 dispersion liquid for specific adsorption. The double-antibody sandwich type immunosensor disclosed by the invention can be used for detecting leather cultural relics, and has a wider linear detection range and a lower detection limit.

Description

Technical Field

[0001] The invention belongs to the technical field of immunosensors, and more specifically relates to a preparation method of an electrochemical immunosensor for detecting leather cultural relics. Background Art

[0002] The main component of leather cultural relics and leather products is collagen. Traditional detection methods include electron microscopic observation methods such as scanning electron microscopy and transmission electron microscopy, and spectral methods such as Fourier infrared spectroscopy and Raman spectroscopy. The material composition is judged based on the observation and absorption characteristic peaks. However, because the samples are precious and have poor recovery, these two methods require multiple detections of different points on the same sample to ensure the reliability of the data. Cultural relics will degrade to a greater or lesser extent after being buried for a long time. For cultural relics that are severely decayed and degraded, their secondary structure is destroyed or even decomposed into peptides, so it is difficult to analyze and detect them using conventional methods. ELISA can also be used for immunological detection of cultural relics, but the detection limit of ELISA is still at the nanogram level, and it is difficult to detect samples with low collagen content or difficult to extract effective information. At present, the combination of immune technology and signal amplification principle can overcome these shortcomings. As a new method, electrochemical immunosensors can play a major role in the detection of cultural relics and leather products, and can perform ultra-sensitive detection of cultural relics.

[0003] Electrochemical immunosensors can be divided into label-free immunosensors and sandwich immunosensors in terms of design. Label-free immunosensors are directly bound to antigens after substrate modification materials and antibodies; sandwich immunosensors are double antibodies sandwiching antigens. Generally speaking, sandwich immunosensors have better performance than label-free immunosensors. The performance of electrochemical immunosensors is closely related to the characteristics of electrodes and their surface modification materials. Glassy carbon electrodes (GCE) are often used as sensor substrates because they are easy to modify. MXene materials, as new two-dimensional materials, can be used as good modification materials combined with glassy carbon electrodes to prepare electrochemical immunosensors because of their high conductivity and flexibility and the presence of functional groups on the surface. However, the easy stacking of MXene two-dimensional materials will affect the performance of the material, which in turn will also affect the performance of electrochemical immunosensors. Summary of the invention

[0004] In view of this, the present invention aims to provide a method for preparing an electrochemical immunosensor for detecting leather cultural relics, successfully prepares a three-dimensional structured MXene / CNTs material and improves the electrochemical properties of the MXene material, combines the bimetallic MIL-101 type MOFs material, and prepares a dual-antibody sandwich immunosensor to detect collagen and leather cultural relics, providing a new technical method for leather cultural relics detection.

[0005] The present invention uses Ti3AlC2 as a raw material, and HCl and LiF as an etching system to selectively etch and intercalate Al in the MAX phase, and then obtains a uniform single-layer MXene dispersion after washing, centrifugation and ultrasound, and uses hexadecyltrimethylammonium bromide (CTAB) to modify CNTs to make them into positively charged and uniformly dispersed CNTs-CTAB, and then the single-layer MXene dispersion and the CNTs-CTAB dispersion are mixed and ultrasound-treated to spontaneously form MXene / CNTs through electrostatic self-assembly, and carbon nanotubes are sandwiched in the middle of MXene in the form of spacers, which can effectively prevent the MXene from restacking, and the two are compositely stacked to form a three-dimensional conductive network structure with higher stability and conductivity.

[0006] At the same time, MIL-101 MOFs were synthesized by solvothermal method using FeCl3 and CoCl2 as raw materials, NH2-BDC as template, and DMF as solvent. MOFs materials are a type of porous, high specific surface area, multi-metal site materials commonly used in catalysis and other applications; MIL-101 is a type of MOFs, which is generated by metal ions provided by metal salts and organic ligands under solvothermal method. The final MOFs can contain functional groups through the selection or functionalization of organic ligands, making them modifiable. Therefore, using NH2-BDC as an organic ligand can make the final MOFs contain -NH2 functional groups. Fe and Co elements have strong catalytic properties. The bimetallic MIL-101 prepared by metal ions provided by metal salts of these two elements has a higher specific surface area, thereby increasing the number of active sites, thereby affecting the amount of loaded antibodies and further affecting the sensitivity of the sensor. By activating the -NH2 on the surface of MOFs with glutaraldehyde fixative GA, it can be covalently coupled to the carboxyl group in streptavidin SA, and then coupled to the biotin-labeled antibody Biotin-Ab2 through the avidin-biotin system. Compared with directly activating -NH2 for connecting antibodies, this method can load more antibodies to improve the sensitivity of the sensor, and finally the sensor's immune probe MOFs-Ab2 is obtained and bound to the substrate.

[0007] The present invention combines two-dimensional MXene with one-dimensional CNTs to form three-dimensional MXene / CNTs, thereby increasing the conductivity of the material and the loadable amount of the active material. The polished GCE is used as the working electrode, Ab1 is the antibody prepared for BLG target 1, and Ab2 is the antibody prepared for BLG target 2. After the MXene / CNTs adsorb the antibody and the antigen BLG, it becomes an immunosensor platform. The prepared bimetallic MOFs can effectively bind to the antibody, and finally complete the preparation of the sandwich immunosensor. After the Ab1 of the substrate binds to BLG or the test object, the sandwich immunosensor will also bind to Biotin-Ab2. In the case of double antibody sandwich, the influence of interference is eliminated to the greatest extent and the antigen-antibody specific binding is performed twice, so it has higher sensitivity, and more importantly, it has stronger specificity and more accurate results.

[0008] In order to achieve the above object, the present invention is implemented by the following technical scheme: a method for preparing an electrochemical immunosensor for detecting leather cultural relics, comprising the following steps:

[0009] Step 1: CNTs are modified with hexadecyltrimethylammonium bromide to obtain a CNTs-CTAB dispersion; the monolayer MXene dispersion and the CNTs-CTAB dispersion are ultrasonically mixed, centrifuged and dried to obtain MXene / CNTs;

[0010] Step 2: Dissolve anhydrous ferric chloride, anhydrous cobalt chloride and aminoterephthalic acid in dimethylformamide respectively, then mix and stir the three solutions evenly, and transfer the mixed solution into a hydrothermal reactor for solvent thermal reaction; after the reaction is completed, wash, centrifuge and vacuum dry to obtain MOFs material;

[0011] Step 3: The MOFs material is redispersed in deionized water, and the resulting MOFs solution is added with glutaraldehyde fixative to react, and then dissolved in PBS buffer after centrifugal washing; the resulting mixed solution a is added with streptavidin solution to react, and then dissolved in PBS buffer after centrifugal washing; the resulting mixed solution b is added with biotin-labeled antibody solution to react, and then washed and centrifuged again to obtain MOFs-Ab2;

[0012] Step 4: Add MXene / CNTs dispersion on the glassy carbon electrode and dry it. Then place it in antibody Ab1 solution to absorb antibodies, and then place it in BSA solution to block the sites. BSA solution is a mixed solution of bovine serum albumin and PBS buffer. Then add leather cultural relic sample solution, and finally place it in MOFs-Ab2 dispersion for specific adsorption.

[0013] Further preferably, in step 1, the preparation of the CNTs-CTAB dispersion comprises: adding CNTs to an aqueous solution of hexadecyltrimethylammonium bromide with a concentration of 0.1-2 mM for ultrasonic dispersion to obtain a CNTs-CTAB dispersion with a CNTs concentration of 0.1-2 wt%.

[0014] Further preferably, in step one, the preparation of the single-layer MXene dispersion includes: taking a hydrochloric acid solution and LiF and placing them in a reaction kettle, adding Ti3AlC2 in small amounts and multiple times, and reacting at 40°C for 18-36 hours; after the reaction is completed, the material is shaken, centrifuged and washed multiple times until the supernatant turns black and the pH of the supernatant is neutral, then the supernatant is ultrasonically layered in an ice-water bath, and finally centrifuged and the supernatant is taken to obtain a single-layer MXene dispersion.

[0015] Further preferably, the concentration of the hydrochloric acid solution is 9-12M; the ratio of the added amounts of the hydrochloric acid solution, LiF and Ti3AlC2 is 20mL:1.5-2g:1g.

[0016] Further preferably, in step 1, the mixing volume ratio of the single-layer MXene dispersion and the CNTs-CTAB dispersion is 2-8:1; and the concentration of the single-layer MXene dispersion is 0.8-1.2 wt%.

[0017] Further preferably, in step 2, the concentrations of the three solutions are the same, all 4-6 mM, and the mixing volume ratio of the three solutions is 1:1:1; the temperature of the solvothermal reaction is 100-120°C, and the reaction time is 20-25h.

[0018] Further preferably, in step three, the mass fraction of the glutaraldehyde fixative is 2.5wt%; and the volume ratio of the glutaraldehyde fixative to the MOFs solution is 1:1-2.

[0019] Further preferably, in step three, the concentration of the streptavidin solution is 1 mg / mL; and the volume ratio of the streptavidin solution to the mixed solution a is 1:3-5.

[0020] Further preferably, in step three, the concentration of the biotin-labeled antibody solution is 100 μg / mL; the volume ratio of the biotin-labeled antibody solution to the mixed solution b is 1:5-8; and the reaction time is 2 h.

[0021] Further preferably, in step 4, the mass fraction of the BSA solution is 1 wt %; the concentration of the antibody Ab1 solution is 3.4 μg / mL; and the concentration of the MOFs-Ab2 dispersion is 5 mg / mL.

[0022] Further preferably, in step 4, the amount of MXene / CNTs dispersion added is 4-8 μL, and the concentration is 1 mg / mL.

[0023] Further preferably, in step 4, the incubation temperature of all assembly steps is 35-40° C., and the incubation time is 0.5-1.5 h.

[0024] Further preferably, in step 4, the amount of the leather cultural relic sample solution added is 10 μL.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) This invention is the first to use the double antibody sandwich method to detect collagen and leather artifacts. The introduction of MXene-based sandwich immunosensor provides a new technical method for leather artifact detection.

[0027] (2) The present invention successfully prepared a three-dimensional MXene / CNTs material and improved the electrochemical properties of the MXene material. This is the first time that this approach has been proposed, applied, and tested in the field of immunosensors.

[0028] (3) The present invention obtains a bimetallic MIL-101 type MOFs material with uniform distribution and full structure by controlling the input concentration of raw materials and controlling the reaction temperature and time, and successfully uses it in the direction of immune sensing.

[0029] (4) Compared with the ELISA method, the immunosensor prepared by the present invention has a wider linear detection range and a lower detection limit, and successfully realizes the detection of cultural relics samples.

[0030] (5) The sensor has good stability, high sensitivity, no hazard, higher reliability and the self-assembly process is easy to characterize, which makes it particularly suitable for detecting collagen and leather artifacts. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the SEM image of MOFs;

[0032] Figure 2 This is the change diagram of the Zeta potential value of the MOFs immune probe;

[0033] Figure 3 is the calibration curve of the EILSA method;

[0034] Figure 4 is the calibration curve (sensitivity) of the immunosensor;

[0035] Figure 5 For specific detection of immunosensors;

[0036] Figure 6To test the stability of the immunosensor;

[0037] Figure 7 Detection of cultural relics samples by immunosensor. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] Example 1

[0040] Step 1: Take 20mL of 9M hydrochloric acid solution and LiF and place them in a reactor and stir at 40℃ for 10min. Add 1g of Ti3AlC2 in small amounts and slowly, and stir the reaction for 24h at 40℃. After the reaction, the material is shaken, centrifuged and washed several times until the supernatant turns black, and the pH of the supernatant is neutral>6. Then, the solution is ultrasonically stratified in an ice-water bath for 5h, and finally centrifuged again. The upper layer is black. The upper layer is the single-layer MXene dispersion, which is stored in a refrigerator at 4℃.

[0041] Step 2: Take a 1 mM aqueous solution of hexadecyltrimethylammonium bromide, add CNTs and perform ultrasonic dispersion to obtain a CNTs-CTAB dispersion with a CNTs mass fraction of 1 wt%.

[0042] Step 3: Take a single-layer MXene solution and dilute it to 1wt%, then mix it with the CNTs-CTAB dispersion at a volume ratio of 6:1 under ultrasound. After mixing and stratification, centrifuge to obtain the precipitate, and vacuum dry the precipitate at 50°C to obtain a MXene / CNTs composite material, which is collected for later use.

[0043] Step 4: Take anhydrous FeCl3, anhydrous CoCl2 and NH2-BDC and dissolve them in DMF respectively. Then, mix the three solutions (all with a concentration of 5mM) at a volume ratio of 1:1:1 for 15 minutes and stir evenly. Transfer the mixed solution into a hydrothermal reactor and perform a solvothermal reaction at 110°C for 24 hours. After the reaction is completed, wash the materials with DMF, ethanol, and deionized water and centrifuge to obtain a brown-yellow precipitate. Then, dry the precipitate in a vacuum drying oven at 60°C for 12 hours to obtain MOFs (MIL-101), which are collected for later use. The SEM image is shown below. Figure 1 shown.

[0044] Step 5: Re-disperse MOFs in deionized water at a concentration of 5 mg / mL, then add it to a 2.5wt% glutaraldehyde fixative at a volume ratio of 1:1 for reaction for 2 hours, then wash with PBS and centrifuge at 3500rpm for 15 minutes, then take the precipitate and redissolve it in PBS to obtain a mixed solution a with a concentration of 5 mg / mL. Take 4 mL of mixed solution a and add it to a 1 mg / mL streptavidin solution at a volume ratio of 4:1, shake it for 2 hours to allow sufficient reaction, wash it again and centrifuge it at 3500rpm for 15 minutes, then redissolve it in PBS to obtain a mixed solution b with a concentration of 5 mg / mL. Add 100 μg / mL of biotin-labeled antibody (Biotin-Ab2) to the mixed solution b, mix them at a volume ratio of 6:1, react for 2 hours, wash it, centrifuge it at 3500rpm for 15 minutes, and redissolve it in PBS to obtain a 5 mg / mL MOFs-Ab2 dispersion, the potential change of which is shown as follows. Figure 2 shown.

[0045] Step 6: Add bovine serum albumin to PBS buffer to obtain a 1wt% BSA solution, which is used as a blocking solution and antibody diluent. A 3mm diameter GCE electrode is polished to a mirror surface in Al2O3 slurry (particle size 50nm), and ultrasonically cleaned in ethanol and deionized water.

[0046] Step 7: Add 6 μL of MXene / CNTs solution with a concentration of 1 mg / mL to the cleaned GCE and dry it, then place it in a 3.4 μg / mL collagen antibody Anti-BLG solution Ab1 to absorb the antibody at 37°C for 1 hour to obtain GCE / MXene / CNTs / Ab1. Then place it in a BSA solution to block the site at 37°C for 1 hour to obtain GCE / MXene / CNTs / Ab1 / BSA. Then add 10 μL of BLG solution with a concentration of 100 pg / mL to 1 mg / mL to obtain GCE / MXene / CNTs / Ab1 / BSA / BLG. Finally, place the sensor in a 5 mg / mL MOFs-Ab2 dispersion at 37°C for 1 hour for specific adsorption to obtain GCE / MXene / CNTs / Ab1 / BSA / BLG / MOFs-Ab2.

[0047] First, the ELISA method was used to detect the binding potency of Ab2 and BLG. The relationship between absorbance and BLG concentration was obtained using an immunoassay kit and absorbance detection. Figure 3 As shown, the method has a good linear relationship when the BLG concentration is 200 ng / mL to 10 μg / mL, and the detection limit is calculated to be 115 ng / mL.

[0048] By calculating the current difference before and after MOFs-Ab2 loading under different BLG concentrations, a calibration curve for immunosensor detection can be made. Figure 4 As shown, there is a good linear relationship in the range of 100 pg / mL to 1 mg / mL, y = 24.481 + 5.62x (R 2 =0.987), and the detection limit can be calculated as 71 pg / mL based on the blank control.

[0049] Example 2 (Specificity)

[0050] Step 1: Take 20mL of 9M hydrochloric acid solution and LiF and place them in a reactor and stir at 40℃ for 10min. Add 1g of Ti3AlC2 in small amounts and slowly, and stir the reaction for 24h at 40℃. After the reaction, the material is shaken, centrifuged and washed several times until the supernatant turns black, and the pH of the supernatant is neutral>6. Then, the solution is ultrasonically stratified in an ice-water bath for 5h, and finally centrifuged again. The upper layer is black. The upper layer is the single-layer MXene dispersion, which is stored in a refrigerator at 4℃.

[0051] Step 2: Take a 1 mM aqueous solution of hexadecyltrimethylammonium bromide, add CNTs and perform ultrasonic dispersion to obtain a CNTs-CTAB dispersion with a CNTs mass fraction of 1 wt%.

[0052] Step 3: Take a single-layer MXene solution and dilute it to 1wt%, then mix it with the CNTs-CTAB dispersion at a volume ratio of 6:1 under ultrasound. After mixing and stratification, centrifuge to obtain the precipitate, and vacuum dry the precipitate at 50°C to obtain a MXene / CNTs composite material, which is collected for later use.

[0053] Step 4: Take anhydrous FeCl3, anhydrous CoCl2 and NH2-BDC and dissolve them in DMF respectively. Then, mix the three solutions (all with a concentration of 5mM) at a volume ratio of 1:1:1 for 15min and stir evenly. Transfer the mixed solution into a hydrothermal reactor and perform a solvothermal reaction at 110°C for 24h. After the reaction is completed, wash the materials with DMF, ethanol, and deionized water and centrifuge to obtain a brown-yellow precipitate. Then, dry the precipitate in a vacuum drying oven at 60°C for 12h to obtain MOFs (MIL-101), which are collected for later use.

[0054] Step 5: Re-disperse MOFs in deionized water at a concentration of 5 mg / mL, then add 2.5wt% glutaraldehyde fixative at a volume ratio of 1:1 to react for 2 hours, then wash with PBS and centrifuge at 3500rpm for 15 minutes to take the precipitate, redissolve in PBS to obtain a mixed solution a with a concentration of 5 mg / mL. Take 4 mL of mixed solution a and add it to 1 mg / mL streptavidin solution at a volume ratio of 4:1, shake for 2 hours to allow sufficient reaction, wash again and centrifuge at 3500rpm for 15 minutes, redissolve in PBS to obtain a mixed solution b with a concentration of 5 mg / mL. Add 100 μg / mL of biotin-labeled antibody (Biotin-Ab2) to mixed solution b, mix at a volume ratio of 6:1, react for 2 hours, wash, centrifuge at 3500rpm for 15 minutes, and redissolve in PBS to obtain a 5 mg / mL MOFs-Ab2 dispersion.

[0055] Step 6: Add bovine serum albumin to PBS buffer to obtain a 1wt% BSA solution, which is used as a blocking solution and antibody diluent. A 3mm diameter GCE electrode is polished to a mirror surface in Al2O3 slurry (particle size 50nm), and ultrasonically cleaned in ethanol and deionized water.

[0056] Step 7: Add 6 μL of MXene / CNTs solution with a concentration of 1 mg / mL to the cleaned GCE and dry it, then place it in a 3.4 μg / mL collagen antibody Anti-BLG solution Ab1 to absorb the antibody at 37°C for 1 hour to obtain GCE / MXene / CNTs / Ab1. Then place it in a BSA solution to block the site at 37°C for 1 hour to obtain GCE / MXene / CNTs / Ab1 / BSA. Then add 10 μL of 1 μg / mL human serum albumin (HSA), ovalbumin (OVA), bovine serum albumin (BSA), porcine collagen (Pig), bovine collagen (Cow), silk fibroin (SF), and wool keratin (WK) as interfering antigen solutions, and antigen BLG as a positive control. Finally, place the sensor in a 5 mg / mL MOFs-Ab2 dispersion at 37°C for 1 hour for specific adsorption.

[0057] Record the current difference before and after MOFs-Ab2 loading, such as Figure 5 As shown, only the positive control group with BLG added was above the detection line, and the other interfering antigens were negative.

[0058] Example 3 (Stability)

[0059] Step 1: Take 20mL of 9M hydrochloric acid solution and LiF and place them in a reactor and stir at 40℃ for 10min. Add 1g of Ti3AlC2 in small amounts and slowly, and stir the reaction for 24h at 40℃. After the reaction, the material is shaken, centrifuged and washed several times until the supernatant turns black, and the pH of the supernatant is neutral>6. Then, the solution is ultrasonically stratified in an ice-water bath for 5h, and finally centrifuged again. The upper layer is black. The upper layer is the single-layer MXene dispersion, which is stored in a refrigerator at 4℃.

[0060] Step 2: Take a 1 mM aqueous solution of hexadecyltrimethylammonium bromide, add CNTs and perform ultrasonic dispersion to obtain a CNTs-CTAB dispersion with a CNTs mass fraction of 1 wt%.

[0061] Step 3: Take a single-layer MXene solution and dilute it to 1wt%, then mix it with the CNTs-CTAB dispersion at a volume ratio of 6:1 under ultrasound. After mixing and stratification, centrifuge to obtain the precipitate, and vacuum dry the precipitate at 50°C to obtain a MXene / CNTs composite material, which is collected for later use.

[0062] Step 4: Take anhydrous FeCl3, anhydrous CoCl2 and NH2-BDC and dissolve them in DMF respectively. Then, mix the three solutions (all with a concentration of 5mM) at a volume ratio of 1:1:1 for 15min and stir evenly. Transfer the mixed solution into a hydrothermal reactor and perform a solvothermal reaction at 110°C for 24h. After the reaction is completed, wash the materials with DMF, ethanol, and deionized water and centrifuge to obtain a brown-yellow precipitate. Then, dry the precipitate in a vacuum drying oven at 60°C for 12h to obtain MOFs (MIL-101), which are collected for later use.

[0063] Step 5: Re-disperse MOFs in deionized water at a concentration of 5 mg / mL, then add 2.5wt% glutaraldehyde fixative at a volume ratio of 1:1 to react for 2 hours, then wash with PBS and centrifuge at 3500rpm for 15 minutes to take the precipitate, redissolve in PBS to obtain a mixture a with a concentration of 5 mg / mL. Take 4 mL of the mixture a and add it to a 1 mg / mL streptavidin solution at a volume ratio of 4:1, shake for 2 hours to allow for sufficient reaction, wash again and centrifuge at 3500rpm for 15 minutes, redissolve in PBS to obtain a mixture b with a concentration of 5 mg / mL. Add 100 μg / mL of biotin-labeled antibody (Biotin-Ab2) to the mixture b, mix at a volume ratio of 6:1, react for 2 hours, wash, centrifuge at 3500rpm for 15 minutes, and redissolve in PBS to obtain a 5 mg / mL MOFs-Ab2 dispersion.

[0064] Step 6: Add bovine serum albumin to PBS buffer to obtain a 1wt% BSA solution, which is used as a blocking solution and antibody diluent. A 3mm diameter GCE electrode is polished to a mirror surface in Al2O3 slurry (particle size 50nm), and ultrasonically cleaned in ethanol and deionized water.

[0065] Step 7: Add 6 μL of 1 mg / mL MXene / CNTs solution to the cleaned GCE and dry it, then place it in 3.4 μg / mL collagen antibody Anti-BLG solution Ab1 to absorb the antibody at 37°C for 1 hour to obtain GCE / MXene / CNTs / Ab1. Then place it in BSA solution to block the site at 37°C for 1 hour to obtain GCE / MXene / CNTs / Ab1 / BSA. Then add 10 μL of 1 μg / mL BLG solution to obtain GCE / MXene / CNTs / Ab1 / BSA / BLG. Finally, place the sensor in 5 mg / mL MOFs-Ab2 dispersion at 37°C for 1 hour for specific adsorption to obtain GCE / MXene / CNTs / Ab1 / BSA / BLG / MOFs-Ab2.

[0066] The prepared sensor was placed in a refrigerator at 4°C for one week, and its peak value was recorded using DPV every day. Figure 6 As shown, the peak current value still remained above 85% of the original electrode after one week, indicating that the sensor has good stability.

[0067] Example 4 (Cultural Relics Sample Detection)

[0068] Step 1: Take 20mL of 9M hydrochloric acid solution and LiF and place them in a reactor and stir at 40℃ for 10min. Add 1g of Ti3AlC2 in small amounts and slowly, and stir the reaction for 24h at 40℃. After the reaction, the material is shaken, centrifuged and washed several times until the supernatant turns black, and the pH of the supernatant is neutral>6. Then, the solution is ultrasonically stratified in an ice-water bath for 5h, and finally centrifuged again. The upper layer is black. The upper layer is the single-layer MXene dispersion, which is stored in a refrigerator at 4℃.

[0069] Step 2: Take a 1 mM aqueous solution of hexadecyltrimethylammonium bromide, add CNTs and perform ultrasonic dispersion to obtain a CNTs-CTAB dispersion with a CNTs mass fraction of 1 wt%.

[0070] Step 3: Take a single-layer MXene solution and dilute it to 1wt%, then mix it with the CNTs-CTAB dispersion at a volume ratio of 6:1 under ultrasound. After mixing and stratification, centrifuge to obtain the precipitate, and vacuum dry the precipitate at 50°C to obtain a MXene / CNTs composite material, which is collected for later use.

[0071] Step 4: Take anhydrous FeCl3, anhydrous CoCl2 and NH2-BDC and dissolve them in DMF respectively. Then, mix the three solutions (all with a concentration of 5mM) at a volume ratio of 1:1:1 for 15min and stir evenly. Transfer the mixed solution into a hydrothermal reactor and perform a solvothermal reaction at 110°C for 24h. After the reaction is completed, wash the materials with DMF, ethanol, and deionized water and centrifuge to obtain a brown-yellow precipitate. Then, dry the precipitate in a vacuum drying oven at 60°C for 12h to obtain MOFs (MIL-101), which are collected for later use.

[0072] Step 5: Re-disperse MOFs in deionized water at a concentration of 5 mg / mL, then add 2.5wt% glutaraldehyde fixative at a volume ratio of 1:1 to react for 2 hours, then wash with PBS and centrifuge at 3500rpm for 15 minutes to take the precipitate, redissolve in PBS to obtain a mixed solution a with a concentration of 5 mg / mL. Take 4 mL of mixed solution a and add it to 1 mg / mL streptavidin solution at a volume ratio of 4:1, shake for 2 hours to allow sufficient reaction, wash again and centrifuge at 3500rpm for 15 minutes, redissolve in PBS to obtain a mixed solution b with a concentration of 5 mg / mL. Add 100 μg / mL of biotin-labeled antibody (Biotin-Ab2) to mixed solution b, mix at a volume ratio of 6:1, react for 2 hours, wash, centrifuge at 3500rpm for 15 minutes, and redissolve in PBS to obtain a 5 mg / mL MOFs-Ab2 dispersion.

[0073] Step 6: Add bovine serum albumin to PBS buffer to obtain a 1wt% BSA solution, which is used as a blocking solution and antibody diluent. A 3mm diameter GCE electrode is polished to a mirror surface in Al2O3 slurry (particle size 50nm), and ultrasonically cleaned in ethanol and deionized water.

[0074] Step 7: Add 6 μL of 1 mg / mL MXene / CNTs solution to the cleaned GCE and dry it, then place it in 3.4 μg / mL collagen antibody Anti-BLG solution Ab1 to absorb the antibody at 37°C for 1 hour to obtain GCE / MXene / CNTs / Ab1. Then place it in BSA solution to block the site at 37°C for 1 hour to obtain GCE / MXene / CNTs / Ab1 / BSA. Place the cultural relic sample (an unknown species taken from the Aisikexiaer Cemetery in Xinjiang) in carbonate buffer and boil it at 98°C for 2 hours to obtain a 1 mg / mL cultural relic sample solution, add 10 μL of it to the electrode surface, and incubate at 37°C for 1 hour. Finally, place the sensor in 5 mg / mL MOFs-Ab2 dispersion at 37°C for 1 hour for specific adsorption.

[0075] The samples can be qualitatively analyzed by calculating the current difference before and after MOFs-Ab2 loading under different samples, such as Figure 7 As shown, among the 6 samples, samples a, c, d, and e were positive above the test line, and samples b and f were negative. BLG is the characteristic sequence extracted from sheep collagen, and Ab1 and Ab2 are two different target-specific antibodies of BLG. Only when the analyte contains sheepskin degradation products can they be effectively loaded onto the immunosensor to obtain a positive result. Therefore, it can be determined that samples a, c, d, and e contain sheepskin.

[0076] The above embodiments of the present invention are merely examples for illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes and modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for preparing an electrochemical immunosensor for detecting leather artifacts, It is characterized in that The steps include: Step 1: CNTs are modified with hexadecyltrimethylammonium bromide to obtain a CNTs-CTAB dispersion; the monolayer MXene dispersion and the CNTs-CTAB dispersion are ultrasonically mixed, centrifuged and dried to obtain MXene / CNTs; Step 2: Dissolve anhydrous ferric chloride, anhydrous cobalt chloride and aminoterephthalic acid in dimethylformamide respectively, then mix and stir the three solutions evenly, and transfer the mixed solution into a hydrothermal reactor for solvent thermal reaction; after the reaction is completed, wash, centrifuge and vacuum dry to obtain MOFs material; Step 3: The MOFs material is redispersed in deionized water, and the resulting MOFs solution is added with glutaraldehyde fixative to react, and then dissolved in PBS buffer after centrifugal washing; the resulting mixed solution a is added with streptavidin solution to react, and then dissolved in PBS buffer after centrifugal washing; the resulting mixed solution b is added with biotin-labeled antibody solution to react, and then washed and centrifuged again to obtain MOFs-Ab2; Step 4: Add MXene / CNTs dispersion on the glassy carbon electrode and dry it. Then place it in antibody Ab1 solution to absorb antibodies, and then place it in BSA solution to block the sites. BSA solution is a mixed solution of bovine serum albumin and PBS buffer. Then add leather cultural relic sample solution, and finally place it in MOFs-Ab2 dispersion for specific adsorption.

2. The method for preparing the electrochemical immunosensor for detecting leather cultural relics as claimed in claim 1, It is characterized in that In step 1, the preparation of the CNTs-CTAB dispersion includes: adding a CNTs dispersion having a mass fraction of 0.1-2 wt % to an aqueous solution of hexadecyltrimethylammonium bromide having a concentration of 0.1-2 mM for ultrasonic dispersion to obtain a CNTs-CTAB dispersion.

3. A method for preparing an electrochemical immunosensor for detecting leather cultural relics as claimed in claim 1 or 2, It is characterized in that In step 1, the preparation of the monolayer MXene dispersion includes: placing a hydrochloric acid solution and LiF in a reaction kettle, adding Ti in small amounts and multiple times 3 AlC 2 , react at 40°C for 18-36h; after the reaction, the material is shaken, centrifuged and washed several times until the supernatant turns black and the pH of the supernatant is neutral, and then the supernatant is ultrasonically stratified in an ice-water bath, and finally centrifuged and the supernatant is taken to obtain a single-layer MXene dispersion.

4. The method for preparing the electrochemical immunosensor for detecting leather cultural relics as claimed in claim 3, It is characterized in that The concentration of the hydrochloric acid solution is 9-12M; The hydrochloric acid solution, LiF and Ti 3 AlC 2 The ratio of added amount is 20mL:1.5-2g:1g.

5. The method for preparing the electrochemical immunosensor for detecting leather cultural relics as claimed in claim 1, It is characterized in that In step 1, the mixing volume ratio of the single-layer MXene dispersion and the CNTs-CTAB dispersion is 2-8:1; and the concentration of the single-layer MXene dispersion is 0.8-1.2 wt%.

6. The method for preparing the electrochemical immunosensor for detecting leather cultural relics as claimed in claim 1, It is characterized in that In step 2, the concentrations of the three solutions are the same, all 4-6 mM, and the mixing volume ratio of the three solutions is 1:1:1; the temperature of the solvothermal reaction is 100-120° C., and the reaction time is 20-25 h.

7. The method for preparing the electrochemical immunosensor for detecting leather cultural relics as claimed in claim 1, It is characterized in that In step 3, the mass fraction of the glutaraldehyde fixative is 2.5wt%; the volume ratio of the glutaraldehyde fixative to the MOFs solution is 1:1-2.

8. The method for preparing the electrochemical immunosensor for detecting leather cultural relics according to claim 1 or 7, It is characterized in that In step 3, the concentration of the streptavidin solution is 1 mg / mL; the volume ratio of the streptavidin solution to the mixed solution a is 1:3-5.

9. The method for preparing the electrochemical immunosensor for detecting leather cultural relics according to claim 1 or 7, It is characterized in that In step 3, the concentration of the biotin-labeled antibody solution is 100 μg / mL; the volume ratio of the biotin-labeled antibody solution to the mixed solution b is 1:5-8; and the reaction time is 2 h.

10. The method for preparing the electrochemical immunosensor for detecting leather cultural relics as claimed in claim 1, It is characterized in that In step 4, the mass fraction of the BSA solution is 1 wt %; the concentration of the antibody Ab1 solution is 3.4 μg / mL; and the concentration of the MOFs-Ab2 dispersion is 5 mg / mL.

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