Preparation method of sandwich structure immunosensor based on surface enhanced Raman scattering and application of sandwich structure immunosensor in detection of anti-phospholipase A2 receptor antibody

Abstract

The invention discloses a preparation method of a sandwich structure immunosensor based on surface enhanced Raman scattering and application of the sandwich structure immunosensor to detection of an anti-phospholipase A2 receptor antibody, and is characterized by comprising the following steps: sputtering silver ions on a cotton swab / ZIF-67 prepared by an in-situ growth method by adopting an ion sputtering method to obtain a cotton swab / ZIF-67 / Ag NPs composite material; the preparation method comprises the following steps: preparing an antigen solution, then adding the antigen solution into the antigen solution to obtain an immune substrate, adding a methylene blue aqueous solution into a silver nanoparticle solution for reaction, then connecting an anti-human PLA2R IgG antibody to obtain an immune probe, and finally dropwise adding a phosphate buffer solution containing an antibody to be detected onto the immune substrate, and dropwise adding the immune probe solution to the immune substrate adsorbed with the antibody to be detected to obtain the sandwich structure immunosensor based on surface enhanced Raman scattering. The method has the advantages of high sensitivity, good selectivity and strong stability.

Description

Technical Field

[0001] The present invention relates to an immunosensor, in particular to a preparation method of a sandwich structure immunosensor based on surface enhanced Raman scattering and its application in anti-phospholipase A2 receptor antibody detection. Background Art

[0002] Surface-Enhanced Raman Scattering (SERS) technology, as a highly sensitive molecular detection method, has been widely used in biomedicine, environmental monitoring, and chemical analysis in recent years. SERS can detect target substances at low concentrations or even single molecule levels by enhancing the Raman signals of molecules on the surface of metal nanostructures. However, traditional SERS substrates usually rely on precious metal (such as gold and silver) nanostructures, which have high preparation costs, poor stability, and are easily disturbed by the environment in practical applications, limiting their large-scale application. Therefore, the development of new, low-cost, and highly stable SERS substrates has become a hot topic in current research.

[0003] In recent years, with the rapid development of nanomaterials and biosensing technology, immunosensors based on nanocomposites have shown great application potential in the field of medical diagnosis. In particular, the combination of metal organic frameworks (MOFs) and noble metal nanoparticles (such as silver nanoparticles, abbreviated as Ag NPs) has been widely used in high-sensitivity and high-selectivity biosensing platforms due to its unique physicochemical properties and biocompatibility. As a typical MOF material, ZIF-67 has a high specific surface area, a tunable pore structure and good chemical stability, which can effectively enhance the signal response of the sensor. At the same time, silver nanoparticles are widely used to improve detection sensitivity due to their excellent optical properties and surface-enhanced Raman scattering effect.

[0004] Phospholipase A2 receptor antibody (abbreviated as PLA2R) is a biomarker closely related to a variety of autoimmune diseases (such as primary membranous nephropathy). Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. About 70%-80% of primary MN (abbreviated as pMN) are associated with PLA2R antibodies. PLA2R is a glycoprotein expressed on the surface of glomerular podocytes. Its autoantibodies (mainly IgG4 subtype) bind to antigens to form immune complexes, which are deposited on the glomerular basement membrane, activate complement and damage the filtration barrier, leading to proteinuria and renal impairment. PLA2R antibody positivity can be used as the gold standard for disease diagnosis.

[0005] The detection methods for PLA2R antibodies include enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IIFT), and Western Blot (WB). WB was the earliest main method for detecting PLA2R antibodies, but it is complicated to operate and has a long cycle, so it is not suitable for clinical application. In 2011, immunofluorescence was introduced into the clinical detection of anti-PLA2R antibodies in serum, but it can only quantify anti-PLA2R antibodies. Currently, the most widely used quantitative detection method for PLA2R antibodies in clinical practice is ELISA, but its detection cost is high, the test results vary greatly, the sensitivity is low, and the linear range is narrow, which limits the clinical detection of PLA2R antibodies to a certain extent. However, there are no relevant research reports on the preparation method of sandwich structure immunosensors based on surface enhanced Raman scattering and their application in the detection of anti-phospholipase A2 receptor antibodies. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing a sandwich structure immunosensor based on surface enhanced Raman scattering with high sensitivity, good selectivity and strong stability, and its application in anti-phospholipase A2 receptor antibody detection.

[0007] The technical solution adopted by the present invention to solve the above technical problems is: a method for preparing a sandwich structure immunosensor based on surface enhanced Raman scattering, comprising the following steps: Step 1: Prepare cotton swab / ZIF-67 by in situ growth method: put the cleaned cotton swab into ZIF-67 precursor methanol solution, react in a 35-40°C oven for 60-150 minutes, take out the cotton swab from the reaction solution, wash it with ethanol, and put the cotton swab into a 35-40°C oven to dry, to obtain a cotton swab / ZIF-67 sample; Step 2: Prepare a cotton swab / ZIF-67 / Ag NPs composite material by ion sputtering: fix the cotton swab / ZIF-67 sample obtained in step 1 and a high-purity silver target in a sputtering chamber, evacuate the chamber to a high vacuum level, and then start the ion sputtering instrument for sputtering for 10-30 seconds, so that silver atoms are sputtered and deposited on the surface of the cotton swab to obtain a cotton swab / ZIF-67 / Ag NPs composite material; Step 3, constructing an immune substrate: adding the cotton swab / ZIF-67 / Ag NPs composite material synthesized in step 2 to 20-200 μL of an antigen solution as a capture molecule, incubating at 4°C for more than 12 h, washing with PBS buffer, blocking with BSA solution, and washing again with PBS buffer to obtain an immune substrate; Step 4, constructing an immune probe: adding a methylene blue aqueous solution to a silver nanoparticle solution, incubating at room temperature and then centrifuging, removing the supernatant, taking the precipitate and resuspending it in a phosphate buffer, and then taking the resuspended solution to connect with an anti-human PLA2R IgG antibody to obtain an immune probe; Step 5, construct a sandwich structure immunosensor based on surface enhanced Raman scattering: add a phosphate buffer solution containing the antibody to be tested to the immune substrate, react at 35-40°C for 1-3 h to allow the immune reaction between the antigen and the antibody to proceed fully, and wash away the excess unreacted antibody to be tested; then add the immune probe solution to the immune substrate adsorbed with the antibody to be tested, react at 35-40°C for 1-3 h, and wash away the excess unreacted immune probe to obtain a sandwich structure immunosensor based on surface enhanced Raman scattering.

[0008] Furthermore, the ZIF-67 precursor methanol solution described in step 1 is prepared by dissolving cobalt nitrate hexahydrate and 2-methylimidazole salt in methanol, the concentration of the cobalt nitrate hexahydrate is 3-42 mg / mL, and the concentration of the 2-methylimidazole salt is 7-28 mg / mL.

[0009] Further, the preparation method of the silver nanoparticle solution described in step 4 is as follows: 0.1-10mL of a concentration of 0.1 A 0.4-0.5 M silver nitrate solution, 3.4-34 mL 0.17 M sodium citrate solution and 0.6-60 mL 0.17 M citric acid solution are mixed and added to 45-450 mL deionized water and stirred evenly, and then 0.2-20 mL 0.5 M sodium borohydride solution is slowly added, and after standing for 5-25 minutes, heated at 100° C. and stirred evenly, and then cooled to room temperature to obtain a silver nanoparticle solution.

[0010] Further, 20-200 μL of 10 -2 M of methylene blue aqueous solution was added to the above silver nanoparticle solution.

[0011] Further, the specific process of connecting the anti-human PLA2R IgG antibody described in step 4 is as follows: 20-200 μL of anti-human PLA2R IgG antibody with a concentration of 0.1 mg / mL is added dropwise to 1-10 mL of the resuspension, incubated at 4°C for 1-3 hours, and then the remaining anti-human PLA2R IgG antibody is removed by centrifugation. The precipitate is resuspended with 200-2000 μL of PBS buffer, and then bovine serum albumin solution is added and incubated at room temperature for 1-3 hours, and then the excess BSA is removed by centrifugation. The precipitate is washed with PBS buffer to obtain an immune probe, and the immune probe is dispersed in 500-1000 μL of PBS buffer to obtain an immune probe solution.

[0012] The present invention also provides a method for detecting anti-phospholipase A2 receptor antibodies using the above-mentioned sandwich structure immunosensor based on surface enhanced Raman scattering. The method is not for the purpose of diagnosis or treatment, and the specific steps are as follows: a phosphate buffer solution containing the anti-phospholipase A2 receptor (PLA2R) antibody to be detected is dripped onto an immune substrate, reacted at 35-40°C for 1-3 h, then 20-200 μL of an immune probe solution is dripped, and the reaction is continued at 35-40°C for 1-3 h. After washing with PBS buffer and drying, a Raman spectrometer is used to measure 1621 cm -1 The Raman signal intensity at wavelength was calculated based on the anti-phospholipase A2 receptor antibody at 1621 cm -1 The linear relationship between the peak intensity of the SERS signal and its concentration was calculated to obtain the concentration of the anti-phospholipase A2 receptor antibody to be tested.

[0013] Compared with the prior art, the advantages of the present invention are: the preparation method of the sandwich structure immunosensor based on surface enhanced Raman scattering and its anti-phospholipase A2 receptor antibody detection application of the present invention, ZIF-67 as a metal organic framework (MOF) material, has a high specific surface area and a controllable pore structure, and can provide more active sites for the specific binding of antigen-antibody. Silver nanoparticles have excellent surface enhanced Raman scattering effect and can significantly enhance the intensity of Raman signals. Through the porous structure of ZIF-67 and the SERS effect of Ag NPs, the sensor can achieve multi-stage amplification of the signal and significantly improve the detection sensitivity. Cotton swabs are used as substrate materials, which are low-cost and easy to obtain. Combined with simple in-situ growth method and ion sputtering technology, large-scale preparation and standardized production can be achieved, reducing the manufacturing cost of the sensor, and are particularly suitable for clinical medical applications. PLA2R antibody and Ag NPs are firmly combined through carbon-nitrogen bonds. Based on the porous structure of ZIF-67 and the SERS effect of Ag NPs, the sensor can achieve ultra-sensitive detection of PLA2R antibody, and the detection limit is significantly lower than that of traditional methods. Through the specific combination of immune probes and immune substrates, the sensor can effectively avoid interference from other biological molecules and ensure high selectivity of the detection results. The entire detection process only takes a few hours, which is much faster than traditional enzyme-linked immunosorbent assay (ELISA) and other time-consuming detection methods, and is suitable for rapid clinical diagnosis.

[0014] In summary, the present invention provides a method for preparing a sandwich structure immunosensor based on surface enhanced Raman scattering and its application in detecting anti-phospholipase A2 receptor antibodies. The sensor synthesizes ZIF-67 on the surface of a cotton swab by an in-situ growth method, and deposits silver nanoparticles on its surface by combining ion sputtering technology to form a composite material with a high specific surface area and excellent optical properties. Subsequently, a sandwich structure is formed by constructing an immune probe and an immune substrate, and the SERS effect of silver nanoparticles is used to achieve high-sensitivity detection of PLA2R antibodies. The sensor not only has the advantages of simple preparation and low cost, but also has high selectivity and stability, providing a new type of rapid detection tool for clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a scanning electron microscope photograph of the cotton swab / ZIF-67 / Ag composite material prepared in Example 1; Figure 2 The SERS spectrum of anti-phospholipase A2 receptor antibody detected by the sandwich structure immunosensor based on surface enhanced Raman scattering prepared in Example 1; Figure 3 is a scanning electron microscope photograph of the cotton swab / ZIF-67 / Ag composite material prepared in Example 2; Figure 4 The SERS spectrum of anti-phospholipase A2 receptor antibody detected by the sandwich structure immunosensor based on surface enhanced Raman scattering prepared in Example 2; Figure 5 This is a scanning electron microscope photograph of the cotton swab / ZIF-67 / Ag composite material prepared in Example 3; Figure 6 The SERS spectrum of anti-phospholipase A2 receptor antibody detected by the sandwich structure immunosensor based on surface enhanced Raman scattering prepared in Example 3; Figure 7 The SERS spectra of the sandwich structure immunosensor based on surface enhanced Raman scattering prepared in Example 1 detecting different concentrations of anti-phospholipase A2 receptor (PLA2R) antibodies; Figure 8 The surface enhanced Raman scattering-based sandwich structure immunosensor prepared in Example 1 detects anti-phospholipase A2 receptor (PLA2R) antibodies at 1621 cm -1 The linear relationship between the peak intensity of SERS signal and its concentration; Fig. 9 The surface enhanced Raman scattering sandwich structure immunosensor prepared in Example 1 detects samples 1-4 at 1621 cm -1 The intensity at (characteristic peak of MB). DETAILED DESCRIPTION

[0016] The present invention is further described in detail below with reference to the accompanying drawings.

[0017] Example 1: A method for preparing a sandwich structure immunosensor based on surface enhanced Raman scattering, comprising the following steps: Step 1: Preparation of cotton swab / ZIF-67 by in situ growth method The cleaned cotton swab was placed in a ZIF-67 precursor methanol solution. After reacting in an oven at 37°C for 100 minutes, the cotton swab was carefully removed from the reaction solution with clean tweezers and thoroughly washed three times with ethanol to remove any unreacted substances and by-products. Each time the cotton swab was washed, it was immersed in 12 mL of ethanol and gently shaken for 12 minutes. After washing, the cotton swab was placed in an oven at 37°C to dry to obtain a cotton swab / ZIF-67 sample. The ZIF-67 precursor methanol solution was prepared by dissolving cobalt nitrate hexahydrate and 2-methylimidazole salt in methanol, wherein cobalt nitrate hexahydrate (Co (NO 3 ) 2 6H 2 O) was 30 mg / mL, and the concentration of 2-methylimidazolium salt was 18 mg / mL.

[0018] Step 2: Preparation of cotton swab / ZIF-67 / Ag NPs composite material by ion sputtering The cotton swab / ZIF-67 sample obtained in step 1 is fixed in a sputtering chamber, and a high-purity silver target is inserted into the sputtering chamber. The chamber is evacuated to a high vacuum level, and then the ion sputtering instrument is turned on for sputtering for 20 seconds. During this process, silver atoms are sputtered from the target and deposited on the surface of the cotton swab to obtain a cotton swab / ZIF-67 / Ag NPs composite material. After the sputtering is completed, the vacuum in the chamber is gradually released, and the cotton swab coated with silver nanoparticles is carefully removed from the sample holder with tweezers and dried and stored; Figure 1 The scanning electron microscope photo of the cotton swab / ZIF-67 / Ag composite material prepared in this example is shown. Figure 1 It can be seen that the prepared cotton swab / ZIF-67 / Ag composite material presents a rough and irregular flower-like shape on the surface of the cotton fiber, and the spherical silver nanoparticles are firmly and evenly anchored on the wrinkled surface of the flower-like ZIF-67 with a smaller size.

[0019] Step 3: Constructing immune matrix The cotton swab / ZIF-67 / Ag NPs composite material prepared in step 2 was added to 100 μL of 0.36 mg / mL PLA2R antigen solution as a capture molecule, and incubated at 4°C for more than 12 h to achieve specific binding of the antigen to the cotton swab / ZIF-67 / AgNPs substrate. It was then washed with PBS buffer to remove unbound antigens, and then 60 μL of 3 wt% BSA solution was added to the substrate for blocking treatment. The substrate was placed at 37°C for 3 h to block nonspecific binding sites. After washing again, the immune substrate was obtained and stored in a dark environment at 4°C.

[0020] Step 4: Construction of immune probe (1) Synthesis of spherical silver nanoparticles: 5 mL of 0.1 20 mL of 0.17 M silver nitrate solution, 20 mL of 0.17 M sodium citrate solution and 30 mL of 0.17 M citric acid solution were mixed and added to 200 mL of deionized water, and placed in a conical flask, stirred at room temperature at a stirring speed of 500 rpm / min for 5 min, and then 10 mL of 0.5 M sodium borohydride solution was slowly added, and after standing for 15 min, the mixture was continuously stirred at a stirring speed of 500 rpm / min for 8 h on a 100 ° C heating plate. After the reaction was completed, a silver nanoparticle solution was obtained, and the solution was allowed to cool to room temperature and transferred to a brown reagent bottle, which was sealed and stored in a 4 ° C refrigerator to prevent oxidation and agglomeration of the silver nanoparticles; (2) Connection of probe molecules: 100 μL of 10 -2 A methylene blue (MB) aqueous solution of 100 μL was added to the silver nanoparticle solution synthesized in step 4 (1), and the mixture was incubated at room temperature for 10 h. The mixture was then centrifuged at 80,000 rpm for 10-20 min, the supernatant was removed, the precipitate was resuspended in 5 mL of phosphate buffered saline (PBS buffer), and the resuspended solution was used for linking with anti-human PLA2R IgG antibody, specifically: 100 μL of 0.1 mg / mL anti-human PLA2R IgG antibody was added dropwise to the resuspended solution, and the mixture was incubated at 4°C for 2 h. The remaining anti-human PLA2R IgG antibody was removed by centrifugation. The precipitate was resuspended in 2000 μL of PBS buffer, and 50 μL of 3 wt% bovine serum albumin (BSA) solution was added to block the inactive sites. The mixture was incubated at room temperature for 1 h, and then the excess BSA was removed by centrifugation. The precipitate was washed with PBS buffer, and the product was dispersed in 8000 μL of PBS buffer to obtain an immune probe solution.

[0021] Step 5: Constructing a sandwich structure immunosensor based on surface enhanced Raman scattering 20 μL of 1× 10 -2The anti-phospholipase A2 receptor (PLA2R) antibody solution of 1000 mg / mL was added to the cotton swab immune substrate obtained in step 3 and incubated at 37°C for 2 h. After the complex was formed by antigen-antibody immune reaction, 20 μL of immune probe solution synthesized in step 4 was added and incubated at 37°C for 2 h. The antibodies were mutually immune recognized and washed 3 times with PBS buffer. The "PLA2R antigen-anti-PLA2R IgG-anti-human IgG" complex was assembled into a sandwich structure and dried. The sample was then immediately measured using a Raman spectrometer. The excitation wavelength of the Raman spectrometer was 532 nm, the laser power was 1 mW, and the acquisition time was 10 seconds. The results are as follows Figure 2 As shown in the SERS spectrum, the detection concentration of the sandwich structure immunosensor based on surface enhanced Raman scattering is 1× 10 -2 The intensity of the anti-phospholipase A2 receptor (PLA2R) antibody solution at 1621 wavenumber (characteristic peak of MB) was 8264.176.

[0022] Example 2: A method for preparing a sandwich structure immunosensor based on surface enhanced Raman scattering, comprising the following steps: Step 1: Preparation of cotton swab / ZIF-67 by in situ growth method The cleaned cotton swab was placed in a ZIF-67 precursor methanol solution. After reacting in an oven at 35°C for 150 min, the cotton swab was carefully removed from the reaction solution with clean tweezers and thoroughly washed with ethanol to remove any unreacted substances and by-products. Each time the cotton swab was washed, it was immersed in 5 mL of ethanol and gently shaken for 5 min. After washing, the cotton swab was placed in an oven at 35°C to dry to obtain a cotton swab / ZIF-67 sample. The ZIF-67 precursor methanol solution was prepared by dissolving cobalt nitrate hexahydrate and 2-methylimidazole salt in methanol, wherein cobalt nitrate hexahydrate (Co (NO 3 ) 2 6H 2 O) was 3 mg / mL, and the concentration of 2-methylimidazole salt was 7 mg / mL.

[0023] Step 2: Preparation of cotton swab / ZIF-67 / Ag NPs composite material by ion sputtering The cotton swab / ZIF-67 sample obtained in step 1 is fixed in a sputtering chamber, and a high-purity silver target is inserted into the sputtering chamber, the chamber is evacuated to a high vacuum level, and then the ion sputtering instrument is turned on for sputtering for 10 seconds to obtain a cotton swab / ZIF-67 / AgNPs composite material. After the sputtering is completed, the vacuum in the chamber is gradually released, and the cotton swab coated with silver nanoparticles is carefully removed from the sample holder with tweezers and dried and stored; Figure 3 The scanning electron microscope photo of the cotton swab / ZIF-67 / Ag composite material prepared in this example is shown. Figure 3 It can be seen that the prepared cotton swab / ZIF-67 / Ag composite material presents a rough and irregular flower-like shape on the surface of the cotton fiber, and the spherical silver nanoparticles are firmly and evenly anchored on the wrinkled surface of the flower-like ZIF-67 with a smaller size.

[0024] Step 3: Constructing immune matrix The cotton swab / ZIF-67 / Ag NPs composite material prepared in step 2 was added to 20 μL of 0.36 mg / mL PLA2R antigen solution as the capture molecule, incubated at 4°C for more than 12 h, and then washed with PBS buffer. Then, 10 μL of 3 wt% BSA solution was added to the substrate for blocking treatment, and the substrate was placed at 37°C for 3 h to block nonspecific binding sites. After washing again, the immune substrate was obtained and stored in a dark environment at 4°C.

[0025] Step 4: Construction of immune probe (1) Synthesis of spherical silver nanoparticles: 0.1 mL of 0.1 3.4 mL of 0.17 M silver nitrate solution, 3.4 mL of 0.17 M sodium citrate solution and 0.60 mL of 0.17 M citric acid solution were mixed and added to 45 mL of deionized water, and placed in a conical flask, stirred at room temperature at a stirring speed of 500 rpm / min for 5 min, and then 0.2 mL of 0.5 M sodium borohydride solution was slowly added, and after standing for 5 min, the mixture was continuously stirred at a stirring speed of 500 rpm / min for 2-12 h on a 100 ° C heating plate. After the reaction was completed, a silver nanoparticle solution was obtained, the solution was allowed to cool to room temperature and the solution was transferred to a brown reagent bottle, which was sealed and stored in a 4 ° C refrigerator to prevent oxidation and agglomeration of the silver nanoparticles; (2) Connection of probe molecules: 20 μL of 10 -2 A methylene blue (MB) aqueous solution of 100 M was added to the silver nanoparticle solution synthesized in step 4 (1), and incubated at room temperature for 2 h. The mixture was then centrifuged at 5000 rpm for 20 min, the supernatant was removed, the precipitate was resuspended in 1 mL of phosphate buffer, and the resuspended solution was used to connect the anti-human PLA2R IgG antibody, specifically: 20 μL of 0.1 mg / mL anti-human PLA2R IgG antibody was added dropwise to the resuspended solution, incubated at 4°C for 1 h, and then the remaining anti-human PLA2R IgG antibody was removed by centrifugation. The precipitate was resuspended in 500 μL of PBS buffer, and 10 uL of 3 wt% bovine serum albumin solution was added to block the inactive sites. After incubation at room temperature for 1 h, the excess BSA was removed by centrifugation. The precipitate was washed with PBS buffer, and the product was dispersed in 500 μL of PBS buffer to obtain an immune probe solution.

[0026] Step 5: Constructing a sandwich structure immunosensor based on surface enhanced Raman scattering 20 μL of 1× 10 -2 Add the anti-phospholipase A2 receptor (PLA2R) antibody solution of 1000 mg / mL to the cotton swab immune substrate obtained in step 3, and incubate at 37°C for 2 h, then drop 20 μL of the immune probe solution synthesized in step 4, and continue incubating at 37°C for 2 h. The antibodies recognize each other, wash three times with PBS buffer and dry, and then immediately measure the sample using a Raman spectrometer. The excitation wavelength of the Raman spectrometer is 532 nm, the laser power is 1 mW, and the acquisition time is 10 seconds. The results are as follows Figure 4 As shown in the SERS spectrum, the detection concentration of the sandwich structure immunosensor based on surface enhanced Raman scattering is 1×10 -2 The intensity of the anti-phospholipase A2 receptor (PLA2R) antibody solution at 1621 wavenumber (characteristic peak of MB) was 8092.46.

[0027] Example 3: A method for preparing a sandwich structure immunosensor based on surface enhanced Raman scattering, comprising the following steps: Step 1: Preparation of cotton swab / ZIF-67 by in situ growth method The cleaned cotton swab was placed in a ZIF-67 precursor methanol solution. After reacting in a 40°C oven for 60 minutes, the cotton swab was carefully removed from the reaction solution with clean tweezers and thoroughly washed with ethanol to remove any unreacted substances and by-products. Each time the cotton swab was washed, it was immersed in 15 mL of ethanol and gently shaken for 15 minutes. After washing, the cotton swab was placed in a 40°C oven to dry to obtain a cotton swab / ZIF-67 sample. The ZIF-67 precursor methanol solution was prepared by dissolving cobalt nitrate hexahydrate and 2-methylimidazole salt in methanol, wherein cobalt nitrate hexahydrate (Co (NO 3 ) 2 6H 2 O) was 42 mg / mL, and the concentration of 2-methylimidazole salt was 28 mg / mL.

[0028] Step 2: Preparation of cotton swab / ZIF-67 / Ag NPs composite material by ion sputtering The cotton swab / ZIF-67 sample obtained in step 1 is fixed in a sputtering chamber, and a high-purity silver target is inserted into the sputtering chamber, the chamber is evacuated to a high vacuum level, and then the ion sputtering instrument is turned on for sputtering for 30 seconds to obtain a cotton swab / ZIF-67 / AgNPs composite material. After the sputtering is completed, the vacuum in the chamber is gradually released, and the cotton swab coated with silver nanoparticles is carefully removed from the sample holder with tweezers and dried and stored; Figure 5The scanning electron microscope photo of the cotton swab / ZIF-67 / Ag composite material prepared in this example is shown. The prepared cotton swab / ZIF-67 / Ag composite material has a rough and irregular flower-like shape on the cotton fiber surface, and the spherical silver nanoparticles are firmly and evenly anchored on the wrinkled surface of the flower-like ZIF-67 with a smaller size.

[0029] Step 3: Constructing immune matrix The cotton swab / ZIF-67 / Ag NPs composite material prepared in step 2 was added to 200 μL of 0.36 mg / mL PLA2R antigen solution as the capture molecule, incubated at 4°C for more than 12 h, and then washed with PBS buffer. Then, 100 μL of 3 wt% BSA solution was added to the substrate for blocking. After being placed at 37°C for 3 h, it was washed again to obtain an immune substrate, which was stored at 4°C in a dark environment.

[0030] Step 4: Construction of immune probe (1) Synthesis of spherical silver nanoparticles: 10 mL of 0.1 34 mL of 0.17 M silver nitrate solution, 34 mL of 0.17 M sodium citrate solution and 60 mL of 0.17 M citric acid solution were mixed and added to 450 mL of deionized water, and placed in a conical flask, stirred at room temperature at a stirring speed of 500 rpm / min for 5 min, and then 20 mL of 0.5 M sodium borohydride solution was slowly added, and after standing for 25 min, the mixture was continuously stirred at a stirring speed of 500 rpm / min for 2-12 h on a 100° C. hot plate. After the reaction was completed, a silver nanoparticle solution was obtained, the solution was allowed to cool to room temperature and the solution was transferred to a brown reagent bottle, which was sealed and stored in a 4° C. refrigerator to prevent oxidation and agglomeration of the silver nanoparticles; (2) Connection of probe molecules: 200 μL of 10 -2 A 100 μL methylene blue aqueous solution was added to the silver nanoparticle solution synthesized in step 4 (1), and the mixture was incubated at room temperature for 24 h. The mixture was then centrifuged at 10,000 rpm for 10 min, the supernatant was removed, the precipitate was resuspended in 10 mL of phosphate buffer, and the resuspended solution was used for connection with anti-human PLA2R IgG antibody, specifically: 200 μL of 0.1 mg / mL anti-human PLA2R IgG antibody was added dropwise to the resuspended solution, and the mixture was incubated at 4°C for 3 h. The remaining anti-human PLA2R IgG antibody was removed by centrifugation. The precipitate was resuspended in 5,000 μL PBS buffer, and 100 μL of 3 wt% bovine serum albumin solution was added to block the inactive sites. The mixture was incubated at room temperature for 1 h, and then the excess BSA was removed by centrifugation. The precipitate was washed with PBS buffer, and the product was dispersed in 1,000 μL PBS buffer to obtain an immune probe solution.

[0031] Step 5: Constructing a sandwich structure immunosensor based on surface enhanced Raman scattering 20 μL of 1× 10 -2 Add 20 μL of anti-phospholipase A2 receptor (PLA2R) antibody solution at 100 mg / mL to the cotton swab immune substrate obtained in step 3 and incubate at 37°C for 2 h. After the complex is formed by antigen-antibody immune reaction, add 20 μL of immune probe solution synthesized in step 4 and continue incubating at 37°C for 2 h. Wash three times with PBS buffer and dry. Then, immediately measure the sample using a Raman spectrometer with an excitation wavelength of 532 nm, a laser power of 1 mW, and an acquisition time of 10 s.

[0032] The results are as follows Figure 6 As shown in the SERS spectrum, the detection concentration of the sandwich structure immunosensor based on surface enhanced Raman scattering is 1× 10 -2 The intensity of the anti-phospholipase A2 receptor (PLA2R) antibody solution at 1621 wavenumber (characteristic peak of MB) was 8109.271.

[0033] Example 4: Sensitivity measurement of sandwich structure immunosensor based on surface enhanced Raman scattering.

[0034] Figure 7 The SERS spectra of the sandwich structure immunosensor based on surface enhanced Raman scattering prepared in Example 1 for detecting anti-phospholipase A2 receptor (PLA2R) antibody. The concentration of anti-phospholipase A2 receptor (PLA2R) antibody increased from 1× 10 -2 mg / mL to 1× 10 -6 mg / mL changes. Figure 7 It can be seen that as the concentration of anti-phospholipase A2 receptor (PLA2R) antibody increases, the SERS signal intensity gradually increases.

[0035] Figure 8 The surface enhanced Raman scattering sandwich structure immunosensor prepared in Example 1 is 1621 cm -1 The corresponding dose response curve of the peak intensity at Figure 8 It can be seen that in 1× 10 -2 mg / mL to 1× 10 -6 In the mg / mL range, the SERS signal intensity showed a good linear relationship with the logarithm of the anti-phospholipase A2 receptor (PLA2R) antibody concentration, and the linear regression equation was y=1764.46x+11427. Based on the three-fold signal-to-noise ratio, the detection limit was estimated to be 3.67× 10 -7mg / mL, indicating that the sandwich structure immunosensor based on surface enhanced Raman scattering can achieve extremely high SERS sensitivity detection.

[0036] Example 5: Application of sandwich structure immunosensor based on surface enhanced Raman scattering.

[0037] Several real human blood samples were collected (obtained from the biobank of the First Affiliated Hospital of Ningbo University with the patient's consent) and centrifuged at 3000 rpm for 20 min at 4°C. The centrifugation was repeated three times to remove residual proteins and other macromolecular components to obtain serum samples, which were then diluted with pH 7.4 phosphate buffer at a ratio of 1:1000 to obtain the test liquid.

[0038] Four of the test liquids were taken out and quantitatively detected by enzyme-linked immunosorbent assay (ELISA) in the clinic, and the titers of PLA2R antibodies in the serum were 25, 40, 67, and 104 RU / mL, respectively, and the clinical test data were arranged from low to high, and marked as samples 1-4. The blocked immune substrate prepared in Example 1 was immersed in the above four test liquids, incubated at 37°C for 1 hour, and captured PLA2R IgG through antigen-antibody specific binding to form a complex, and then washed 3 times with PBS buffer, and then 20 μL of the immune probe solution prepared in Example 1 was dripped, and continued to incubate at 37°C for 2 hours to form a complete sandwich structure (PLA2R antigen-PLA2R IgG of the patient to be tested-probe secondary antibody), and then washed 3 times with PBS buffer and dried. On this basis, the sample was immediately measured using a Raman spectrometer, the excitation wavelength of the Raman spectrometer was 532 nanometers, the laser power was 1 milliwatt, and the acquisition time was 10 seconds. Fig. 9 The surface enhanced Raman scattering sandwich structure immunosensor prepared in Example 1 detects samples 1-4 at 1621 cm -1 The intensities at (characteristic peaks of MB) are 2483.8674, 3404.0232, 4230.2715, and 6152.5649, respectively. Fig. 9 It can be seen that the SERS signal intensity of samples 1-4 is gradually increasing, and the results of SERS detection and clinical detection show a consistent concentration gradient trend between samples. The rules of SERS detection and clinical detection are basically consistent.

[0039] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A method for preparing a sandwich structure immunosensor based on surface enhanced Raman scattering, characterized in that The following steps are involved: Step 1, using an in-situ growth method, placing a cotton swab into a ZIF-67 precursor methanol solution to prepare a cotton swab / ZIF-67; Step 2, using an ion sputtering method to sputter and deposit silver atoms on the surface of a cotton swab to prepare a cotton swab / ZIF-67 / Ag NPs composite material; Step 3, adding the cotton swab / ZIF-67 / Ag NPs composite material synthesized in step 2 to the antigen solution as the capture molecule, and incubating to obtain the immune substrate; Step 4, adding a methylene blue aqueous solution to the silver nanoparticle solution, incubating at room temperature and then centrifuging, taking the precipitate and resuspending it in phosphate buffer, and then taking the resuspended solution to connect with anti-human PLA2R IgG antibody to obtain an immune probe solution; Step 5: After the phosphate buffer solution containing the antibody to be tested is added dropwise to the immune substrate for reaction, the immune probe solution is then added dropwise to the immune substrate adsorbed with the antibody to be tested for reaction, thereby obtaining a sandwich structure immunosensor based on surface enhanced Raman scattering.

2. The method for preparing the sandwich structure immunosensor based on surface enhanced Raman scattering according to claim 1, characterized in that Step 1 is as follows: put the cleaned cotton swab into the ZIF-67 precursor methanol solution, react in a 35-40°C oven for 60-150 minutes, take the cotton swab out of the reaction solution, wash it with ethanol, and put the cotton swab in a 35-40°C oven to dry to obtain a cotton swab / ZIF-67 sample.

3. The method for preparing the sandwich structure immunosensor based on surface enhanced Raman scattering according to claim 2, characterized in that: The ZIF-67 precursor methanol solution described in step 1 is prepared by dissolving cobalt nitrate hexahydrate and 2-methylimidazole salt in methanol, wherein the concentration of the cobalt nitrate hexahydrate is 3-42 mg / mL, and the concentration of the 2-methylimidazole salt is 7-28 mg / mL.

4. The method for preparing the sandwich structure immunosensor based on surface enhanced Raman scattering according to claim 1, characterized in that The preparation method of the silver nanoparticle solution described in step 4 is as follows: 0.1-10 mL of a 0.1 M silver nitrate solution, 3.4-34 mL of a 0.17 M sodium citrate solution and 0.6-60 mL of a 0.17 M citric acid solution are mixed and added to 45-450 mL of deionized water and stirred evenly, and then 0.2-20 mL of a 0.5 M sodium borohydride solution is slowly added, and after standing for 5-25 minutes, the mixture is heated at 100° C. and stirred evenly, and then cooled to room temperature to obtain a silver nanoparticle solution.

5. The method for preparing the sandwich structure immunosensor based on surface enhanced Raman scattering according to claim 4, characterized in that: 20-200 μL of 10 -2 M of methylene blue aqueous solution was added to the silver nanoparticle solution.

6. The method for preparing the sandwich structure immunosensor based on surface enhanced Raman scattering according to claim 1, characterized in that The specific process of connecting the anti-human PLA2R IgG antibody described in step 4 is as follows: 20-200 μL of anti-human PLA2R IgG antibody with a concentration of 0.1 mg / mL is added dropwise to 1-10 mL of the resuspension solution, incubated at 4°C for 1-3 hours, and then the remaining anti-human PLA2R IgG antibody is removed by centrifugation. The precipitate is resuspended with 500-5000 μL of PBS buffer, and then bovine serum albumin solution is added and incubated at room temperature for 1-3 hours, and then the excess BSA is removed by centrifugation. The precipitate is washed with PBS buffer to obtain an immune probe, and the immune probe is dispersed in 500-1000 μL of PBS buffer to obtain an immune probe solution.

7. The method for preparing the sandwich structure immunosensor based on surface enhanced Raman scattering according to claim 1, characterized in that Step 5 is specifically as follows: a phosphate buffer solution containing the antibody to be tested is added dropwise to the immune substrate, reacted at 35-40°C for 1-3 h, and the excess unreacted antibody to be tested is removed by washing; then, an immune probe solution is added dropwise to the immune substrate adsorbed with the antibody to be tested, reacted at 35-40°C for 1-3 h, and the excess unreacted immune probe is removed by washing, thereby obtaining a sandwich structure immunosensor based on surface enhanced Raman scattering.

8. A method for detecting anti-phospholipase A2 receptor antibodies using a sandwich structure immunosensor based on surface enhanced Raman scattering as described in any one of claims 1 to 7, wherein the method is not for the purpose of diagnosis or treatment, and is characterized in that The specific steps are as follows: add a phosphate buffer solution containing the anti-phospholipase A2 receptor antibody to be tested onto the immune substrate, react at 35-40°C for 1-3 h, then add 20-200 μL of the immune probe solution, continue to react at 35-40°C for 1-3 h, wash with PBS buffer and dry, and use a Raman spectrometer to measure 1621 cm -1 The Raman signal intensity at wavelength was calculated based on the anti-phospholipase A2 receptor antibody at 1621 cm -1 The linear relationship between the peak intensity of the SERS signal and its concentration was calculated to obtain the concentration of the anti-phospholipase A2 receptor antibody to be tested.

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