Preparation method of g-C3N4 / ZIF-67 composite surface enhanced Raman scattering substrate and application of g-C3N4 / ZIF-67 composite surface enhanced Raman scattering substrate in A beta protein detection

Abstract

The invention discloses a preparation method of a g-C3N4 / ZIF-67 composite surface enhanced Raman scattering substrate and Abeta protein detection application of the g-C3N4 / ZIF-67 composite surface enhanced Raman scattering substrate. The preparation method is characterized by comprising the following steps: performing ultrasonic dispersion on a PEI functionalized g-C3N4 nanosheet in ethanol, and then adding AlCl3. 6H2O to obtain a g-C3N4 nanosheet suspension; the preparation method of the g-C3N4 / ZIF-67 composite surface enhanced Raman scattering substrate comprises the following steps: putting a cleaned copper foil and a platinum sheet into a g-C3N4 nanosheet suspension, thoroughly washing the copper foil with ethanol after electro-deposition, carrying out vacuum drying to obtain a g-C3N4 nanosheet / copper foil, and finally modifying the g-C3N4 nanosheet / copper foil with ZIF-67 through secondary electro-deposition to prepare the g-C3N4 / ZIF-67 composite surface enhanced Raman scattering substrate. High-sensitivity detection of biomolecules such as A beta protein is realized, and the preparation method is simple, efficient and suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate, and in particular to a preparation method of a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate and its application in detecting Aβ protein. Background Technique

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

[0003] Graphitic carbon nitride (abbreviated as g-C3N4), as a non-metallic semiconductor material, has excellent optical properties, chemical stability and biocompatibility, and has shown great potential in the fields of photocatalysis and sensing in recent years. Especially g-C3N4 nanosheets, due to their large specific surface area and abundant surface functional groups, can effectively adsorb target molecules and are ideal candidate materials for SERS substrates. In addition, Metal-Organic Frameworks (abbreviated as MOF), due to their highly ordered porous structure and tunable chemical properties, can further enhance the adsorption capacity and signal enhancement effect of SERS substrates. However, the preparation of existing g-CN / ZIF-67 composites usually involves multiple-step reactions and strict condition control, which increases the difficulty and cost of large-scale production. At the same time, although ZIF-67 itself has a high specific surface area and porous structure, after being combined with g-CN, the coverage of g-CN may partially block the pores of ZIF-67, resulting in a decrease in the specific surface area and porosity of the composite material. This will directly affect the adsorption ability of the material for the molecule to be detected, and further affect its signal enhancement effect in Raman detection. In addition, the interfacial combination of g-CN and ZIF-67 may not be ideal, resulting in a decrease in electron transfer efficiency. Due to the large differences in chemical properties and structures between g-CN and ZIF-67, the interfacial combination between the two may not be tight enough, affecting charge transfer and chemical enhancement effects. This will weaken the signal enhancement ability of the composite material in Raman detection and reduce the detection sensitivity. In addition, the existing preparation methods are difficult to precisely control the distribution and density of g-CN and ZIF-67 in the composite material, resulting in poor uniformity and controllability of the material. This will directly affect the Raman enhancement effect of the composite material, especially in applications that require highly reproducible detection, and the performance of the material may not meet the requirements.

[0004] The Aβ protein (Amyloid-β protein) is a polypeptide fragment produced by the amyloid precursor protein (APP) through enzymatic cleavage, mainly composed of 40 or 42 amino acids (referred to as Aβ40 and Aβ42 respectively). The Aβ protein plays a key role in the pathogenesis of Alzheimer's disease (abbreviated as AD). The abnormal aggregation and deposition of the Aβ protein form amyloid plaques, which are considered to be one of the main causes of neuronal damage and cognitive decline. Therefore, the detection of the Aβ protein is of great significance for the early diagnosis and pathological research of Alzheimer's disease.

[0005] Existing Aβ protein detection methods include enzyme-linked immunosorbent assay (ELISA), Western blot, and mass spectrometry (MS). Although they have played an important role in the research and diagnosis of Alzheimer's disease, there are still problems such as limited sensitivity, complex operation, expensive equipment, and cumbersome sample processing. These disadvantages limit the wide application of Aβ protein detection, especially in early diagnosis and low-concentration detection. Therefore, developing a highly sensitive, highly selective, simple-to-operate, and low-cost Aβ protein detection method has important scientific significance and clinical application value. However, there is no publicly reported research on the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate and its application in Aβ protein screening. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a preparation method of a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate with significantly enhanced Raman signals and improved detection sensitivity, as well as its application in Aβ protein detection.

[0007] The technical solution adopted by the present invention to solve the above technical problems is as follows: A preparation method of a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate, which is characterized by including the following steps: Step 1: Ultrasonically disperse PEI-functionalized g-C3N4 nanosheets in ethanol for 0.5 - 2 hours, then add AlCl3·6H2O to obtain a g-C3N4 nanosheet suspension; place the cleaned copper foil and platinum sheet in the g-C3N4 nanosheet suspension, after electrodeposition, thoroughly rinse the copper foil with ethanol and then dry it in vacuum to obtain g-C3N4 nanosheet / copper foil, where the mixing ratio of PEI-functionalized g-C3N4 nanosheets, AlCl3·6H2O, and ethanol is: 5 mg : 1 - 3 mg : 10 - 30 mL; Step 2: Prepare a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate by modifying flower-like ZIF-67 on the g-C3N4 nanosheet / copper foil through secondary electrodeposition.

[0008] Furthermore, the synthesis of the PEI-functionalized g-C3N4 nanosheets in Step 1 includes the following steps: Step 1-1, preparation of g-C3N4 nanosheets: dissolve bulk g-C3N4 in concentrated hydrochloric acid, stir evenly to obtain a g-C3N4 suspension with a concentration of 15-20mM, perform ultrasonic treatment until its color changes from light green to milky white, transfer the milky white suspension to a polytetrafluoroethylene container, react at 70-90°C for 12-24 hours, centrifuge to obtain the supernatant, wash the precipitate with deionized water and then centrifuge to obtain the supernatant, after multiple centrifugation and washing, collect the supernatant obtained from each centrifugation to obtain the g-C3N4 supernatant; Step 1-2, add 20-30 mg / mL polyethyleneimine (abbreviated as PEI) aqueous solution dropwise to the g-C3N4 supernatant obtained in step 1, and after fully mixing under ultrasonic treatment, the mixture is continuously stirred at a rate of 120-180 rpm for 12-24 hours, centrifuged to obtain the supernatant, the precipitate is washed with deionized water and then centrifuged to obtain the supernatant, after multiple centrifugation and washing, the supernatant obtained from each centrifugation is collected and vacuum dried to obtain PEI functionalized g-C3N4 nanosheets, wherein the mass ratio of the polyethyleneimine aqueous solution to the g-C3N4 supernatant is controlled to be 2:1.

[0009] Furthermore, in step 1, during the electrodeposition process, the distance between the two electrodes is 2-4 cm, the deposition time is controlled to be 60-240 seconds, and the deposition voltage is 15-24V.

[0010] Furthermore, step 2 is specifically as follows: using a two-electrode system, the g-C3N4 nanosheet / copper foil prepared in step 1 is used as the negative electrode and the platinum sheet is used as the positive electrode, and is placed in a flower-shaped ZIF-67 solution with a concentration of 10-20 mmol / mL, and the deposition voltage is adjusted to 6-12 V within a constant time of 300 seconds, and flower-shaped ZIF-67 with different densities is grown on the surface of the g-C3N4 nanosheet / copper foil, which is washed and dried to obtain a g-C3N4 / ZIF-67 composite surface enhanced Raman scattering substrate.

[0011] Furthermore, the preparation of the flower-shaped ZIF-67 solution includes the following steps: using methanol to prepare a 2-methylimidazole solution with a concentration of 14-140 mg / mL and a Co(NO3)2·6H2O solution with a concentration of 21-210 mg / mL, respectively; after fully mixing the 2-methylimidazole solution and the Co(NO3)2·6H2O solution in a volume ratio of 1:3, incubating at room temperature for 1-3 hours, washing with ethanol several times, and obtaining a flower-shaped ZIF-67 solution with a concentration of 10-20 mmol / mL.

[0012] The present invention also provides an application of the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared by the above method in the detection of Aβ protein. This method is not for diagnosis or treatment purposes, and is characterized in that: the Aβ protein solution to be detected is modified on the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate. After incubation for 4-8 hours, the sample is immediately measured using a Raman microscope. According to the linear relationship between the Raman intensity and the concentration of the Aβ protein solution, the concentration of the Aβ protein in the Aβ protein solution to be detected is calculated.

[0013] The present invention also provides an application of the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared by the above method in the preparation of a diagnostic reagent for Alzheimer's disease.

[0014] Compared with the prior art, the advantages of the present invention are as follows: For the preparation method of the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate and its application in Aβ protein detection of the present invention, the g-C3N4 nanosheets have a large specific surface area and rich surface functional groups, which can effectively adsorb target molecules and enhance the intensity of the SERS signal. Through functionalization with polyethyleneimine (PEI), the surface of the g-C3N4 nanosheets is positively charged, and it can adsorb ZIF-67 with surface ligands through electrostatic interaction to form a stable composite structure. ZIF-67 is a metal-organic framework material with a highly ordered porous structure. The unique morphology of its flower-like ZIF-67 further increases the specific surface area of the substrate, provides more adsorption sites, can significantly improve the adsorption efficiency of the protein molecules to be detected, and enhance the detection sensitivity of the SERS substrate. Further, through the effective composite of g-C3N4 and ZIF-67, a semiconductor heterojunction can be formed, further promoting the charge transfer efficiency between the two, causing a synergistic chemical enhancement effect. The substrate can significantly enhance the Raman signal and achieve high-sensitivity detection of low-concentration target molecules. At the same time, through the electrodeposition process, the distribution and density of g-C3N4 nanosheets and ZIF-67 on the substrate surface can be precisely controlled, so that the g-C3N4 nanosheets form a uniform and dense coating on the copper foil surface, and the flower-like ZIF-67 also forms a relatively uniform distribution, significantly improving the substrate uniformity and the repeatability of the SERS signal. In particular, the uniform and dense coating of the g-C3N4 nanosheets enables them to form a more stable and effective contact composite with the flower-like ZIF-67, improving the charge transfer efficiency of the composite material, being conducive to charge transfer and chemical enhancement effects, and significantly increasing the enhancement degree of the SERS signal.

[0015] In summary, the present invention relates to a method for preparing a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate and its application in detecting Aβ protein. By optimizing the preparation, functionalization of g-C3N4 nanosheets and the electrodeposition composite process with ZIF-67, high-sensitivity detection of biomolecules such as Aβ protein is achieved. The preparation method is simple, efficient and suitable for large-scale production. This technology not only provides a new idea for the development of SERS substrates, but also offers a potential solution for the early diagnosis of neurodegenerative diseases such as Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 FIG. is a scanning electron microscope photograph of the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared in Example 1; Figure 2 FIG. is a SERS spectrum of Aβ42 detected using the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared in Example 1; Figure 3 FIG. is a scanning electron microscope photograph of the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared in Example 2; Figure 4 FIG. is a SERS spectrum of Aβ42 detected using the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared in Example 2; Figure 5 FIG. is a scanning electron microscope photograph of the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared in Example 3; Figure 6 FIG. is a SERS spectrum of different concentrations of Aβ42 detected using the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared in Example 3; Figure 7 FIG. is a SERS spectrum of different concentrations of Aβ42 detected using the composite surface-enhanced Raman scattering substrate prepared in Example 2; Figure 8 FIG. shows the linear relationship between the SERS signal peak intensity of Aβ42 detected by the composite surface-enhanced Raman scattering substrate prepared in Example 2 at 1365 cm -1 and its concentration; Figure 9 FIG. is a SERS spectrum of Aβ42 detected using the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared in Example 5. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

[0018] Embodiment 1: A method for preparing a g-C3N4 / ZIF-67 composite surface enhanced Raman scattering substrate, comprising the following steps: Step 1: Preparation of g-C3N4 nanosheets The g-C3N4 nanosheets were prepared by a typical acid etching method, as follows: bulk g-C3N4 was dissolved in concentrated hydrochloric acid, stirred evenly to obtain a g-C3N4 suspension with a concentration of 15 mM, and then subjected to ultrasonic treatment until its color changed from light green to milky white, the milky white suspension was transferred to a polytetrafluoroethylene container, reacted at 70°C for 24 hours, and then centrifuged to obtain a supernatant, the precipitate was washed with deionized water, and then centrifuged to obtain a supernatant, and after multiple centrifugation and washing, the supernatant obtained from each centrifugation was collected to obtain a g-C3N4 supernatant; Step 2: Synthesis of PEI functionalized g-C3N4 nanosheets PEI functionalized g-C3N4 nanosheets were prepared by a simple electrostatic self-assembly strategy: 25 mg / mL PEI aqueous solution was added dropwise to the g-C3N4 supernatant obtained in step 1 at a mass ratio of 2:1, and the mixture was fully mixed under ultrasonic treatment. After continuous stirring at a rate of 120 rpm for 24 hours, the supernatant was centrifuged to obtain the supernatant, the precipitate was washed with deionized water and then centrifuged to obtain the supernatant, and after multiple centrifugation and washing, the supernatant obtained from each centrifugation was collected and dried in a vacuum oven at 60°C for 12 hours to obtain PEI functionalized g-C3N4 nanosheets; Step 3: Preparation of flower-shaped ZIF-67 First, methanol was used to prepare a 14 mg / mL 2-methylimidazole solution and a 21 mg / mL Co(NO3)2·6H2O solution, respectively. The 2-methylimidazole solution and the Co(NO3)2·6H2O solution were fully mixed at a volume ratio of 1:3, incubated at room temperature for 2 hours, and then washed three times with ethanol to obtain a 10 mmol / mL flower-shaped ZIF-67 solution. Step 4: Electrodeposition of g-C3N4 film First, a copper foil with a thickness of 1 mm was cut into a size of 5 mm × 10 mm and cleaned with acetone, ethanol and deionized water for later use; After ultrasonically dispersing 5 mg of the PEI-functionalized g-C3N4 nanosheets prepared in Step 2 in 10 mL of ethanol for 0.5 hour, 1 mg of AlCl3·6H2O was added to promote the deposition of the g-C3N4 nanosheets, obtaining a g-C3N4 nanosheet suspension; the cleaned copper foil and a platinum sheet of the same size were respectively used as the electrodes for the electrodeposition experiment and placed in the g-C3N4 nanosheet suspension. The distance between the two electrodes was controlled to be 2 cm, the deposition time was 60 seconds, and the deposition voltage was 15 V. The positively charged g-C3N4 nanosheets moved towards the copper foil electrode and finally deposited on its surface. The copper foil was thoroughly rinsed three times with ethanol and then dried in a vacuum oven at 45 °C for 1 hour to obtain g-C3N4 nanosheet / copper foil; Step 5. Preparation of a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate by secondary electrodeposition. The g-C3N4 / ZIF-67 composite substrate was prepared by modifying ZIF-67 on the g-C3N4 nanosheet / copper foil through a secondary electrodeposition process. Using a two-electrode system, the g-C3N4 nanosheet / copper foil prepared in Step 4 was used as the negative electrode, and the platinum sheet was used as the positive electrode and placed in a flower-like ZIF-67 solution with a concentration of 10 mmol / mL. By adjusting the deposition voltage to 8 V within a constant time of 300 seconds, flower-like ZIF-67 with different densities grew on the surface of the g-C3N4 nanosheet / copper foil. After washing and drying, a surface-enhanced Raman scattering substrate of the g-C3N4 / ZIF-67 composite material was obtained.

[0019] Figure 1 Showing the scanning electron microscope photograph of the g-C3N4 / ZIF-67 composite material prepared in this example. As can be seen from Figure 1 it, the g-C3N4 nanosheets are densely coated on the copper foil, and ZIF-67 microflowers are scattered on it, forming a good composite of the two.

[0020] A 1.0×10 -4 M Aβ42 standard solution was dropped on the surface-enhanced Raman scattering substrate of the g-C3N4 / ZIF-67 composite material and incubated for 6 hours. Finally, the sample was immediately measured using a Raman microscope. The excitation wavelength of the Raman spectrometer was 532 nm, the laser power was 1 mW, and the acquisition time was 10 seconds. It should be noted that all SERS spectra are the average values of 5 sets of data to ensure the reliability of the results. Figure 2 The SERS spectrum for detecting Aβ42 using the surface-enhanced Raman scattering substrate of the g-C3N4 / ZIF-67 composite material prepared in this example. As can be seen from Figure 2 it, the intensity of Aβ42 at 1365 wavenumbers is 573.

[0021] Example 2. A preparation method of a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate, comprising the following steps: Step 1: Preparation of g-C3N4 nanosheets The g-C3N4 nanosheets were prepared by a typical acid etching method, as follows: bulk g-C3N4 was dissolved in concentrated hydrochloric acid, stirred evenly to obtain a g-C3N4 suspension with a concentration of 18 mM, and then subjected to ultrasonic treatment until its color changed from light green to milky white, the milky white suspension was transferred to a polytetrafluoroethylene container, reacted at 80°C for 18 hours, and then centrifuged to obtain a supernatant, the precipitate was washed with deionized water, and then centrifuged to obtain a supernatant, and after multiple centrifugation and washing, the supernatant obtained from each centrifugation was collected to obtain a g-C3N4 supernatant; Step 2: Synthesis of PEI functionalized g-C3N4 nanosheets PEI functionalized g-C3N4 nanosheets were prepared by a simple electrostatic self-assembly strategy: 125 mg / mL PEI aqueous solution was added dropwise to the g-C3N4 supernatant obtained in step 1 at a mass ratio of 2:1, and the mixture was fully mixed under ultrasonic treatment. After continuous stirring at a rate of 150 rpm for 18 hours, the supernatant was obtained by centrifugation, the precipitate was washed with deionized water and then centrifuged to obtain the supernatant. After multiple centrifugations and washings, the supernatants obtained from each centrifugation were collected and dried in a vacuum oven at 60°C for 12 hours to obtain PEI functionalized g-C3N4 nanosheets. Step 3: Preparation of flower-shaped ZIF-67 First, methanol was used to prepare a 70 mg / mL 2-methylimidazole solution and a 105 mg / mL Co(NO3)2·6H2O solution, respectively. The 2-methylimidazole solution and the Co(NO3)2·6H2O solution were fully mixed at a volume ratio of 1:3, incubated at room temperature for 2 hours, and then washed three times with ethanol to obtain a 15 mmol / mL flower-shaped ZIF-67 solution. Step 4: Electrodeposition of g-C3N4 film First, a copper foil with a thickness of 1 mm was cut into a size of 5 mm × 10 mm and cleaned with acetone, ethanol and deionized water for later use; After 5 mg of the PEI-functionalized g-C3N4 nanosheets prepared in step 2 were ultrasonically dispersed in 20 mL of ethanol for 1 hour, 2 mg of AlCl3·6H2O was added to promote the deposition of g-C3N4 nanosheets to obtain a g-C3N4 nanosheet suspension; The cleaned copper foil and platinum sheet of the same size were placed in the g-C3N4 nanosheet suspension as electrodes for the electrodeposition experiment, respectively. The distance between the two electrodes was controlled to be 3 cm, the deposition time was 120 seconds, and the deposition voltage was 20 V. The positively charged g-C3N4 nanosheet moved toward the copper foil electrode and finally deposited on its surface. The copper foil was thoroughly rinsed with ethanol three times and then dried in a vacuum oven at 45 ° C for 1 hour to obtain g-C3N4 nanosheet / copper foil. Step 5: Preparation of g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate. The g-C3N4 / ZIF-67 composite substrate is prepared by modifying ZIF-67 on g-C3N4 nanosheets / copper foil through a secondary electrodeposition process. Using a two-electrode system, the g-C3N4 nanosheets / copper foil prepared in Step 4 is used as the negative electrode, and a platinum sheet is used as the positive electrode. They are placed in a flower-like ZIF-67 solution with a concentration of 15 mmol / mL. By adjusting the deposition voltage to 8 V within a constant time of 300 seconds, flower-like ZIF-67 with different densities grows on the surface of the g-C3N4 nanosheets / copper foil. After washing and drying, a g-C3N4 / ZIF-67 composite material surface-enhanced Raman scattering substrate is obtained.

[0022] Figure 3 Shows the scanning electron microscope photograph of the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared in this example. As can be seen from Figure 3 it, the g-C3N4 nanosheets are densely coated on the copper foil, and micron-sized flowers of ZIF-67 are scattered on it, forming a good composite of the two.

[0023] A 1.0×10 -4 M Aβ42 standard solution is dropped on the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate and incubated for 6 hours. Finally, the sample is immediately measured using a Raman microscope. The excitation wavelength of the Raman spectrometer is 532 nm, the laser power is 1 mW, and the acquisition time is 10 seconds. Figure 4 Is the SERS spectrum of Aβ42 detected using the g-C3N4 / ZIF-67 composite material surface-enhanced Raman scattering substrate prepared in this example. As can be seen from Figure 4 it, the intensity of Aβ42 at a wavenumber of 1365 is 632.

[0024] Example 3: A preparation method of a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate, comprising the following steps: Step 1: Preparation of g-C3N4 nanosheets The g-C3N4 nanosheets are prepared by a typical acid etching method, specifically as follows: The bulk g-C3N4 is dissolved in concentrated hydrochloric acid, and after stirring evenly to obtain a g-C3N4 suspension with a concentration of 24 mM, ultrasonic treatment is carried out until its color changes from light green to milky white. The milky white suspension is transferred to a polytetrafluoroethylene container and reacted at 90 °C for 12 hours. Then, the supernatant is taken by centrifugation. The precipitate is washed with deionized water and then the supernatant is taken by centrifugation again. After multiple centrifugations and washings, the supernatants obtained from each centrifugation are collected to obtain the g-C3N4 supernatant; Step 2: Synthesis of PEI-functionalized g-C3N4 nanosheets PEI functionalized g-C3N4 nanosheets were prepared by a simple electrostatic self-assembly strategy: 250 mg / mL PEI aqueous solution was added dropwise to the g-C3N4 supernatant obtained in step 1 at a mass ratio of 2:1, and the mixture was fully mixed under ultrasonic treatment. After continuous stirring at a rate of 180 rpm for 12 hours, the supernatant was centrifuged to obtain the supernatant, the precipitate was washed with deionized water and then centrifuged to obtain the supernatant, and after multiple centrifugation and washing, the supernatant obtained from each centrifugation was collected and dried in a vacuum oven at 60°C for 12 hours to obtain PEI functionalized g-C3N4 nanosheets; Step 3: Preparation of flower-shaped ZIF-67 First, methanol was used to prepare a 140 mg / mL 2-methylimidazole solution and a 210 mg / mL Co(NO3)2·6H2O solution, respectively. The 2-methylimidazole solution and the Co(NO3)2·6H2O solution were fully mixed at a volume ratio of 1:3, incubated at room temperature for 2 hours, and then washed three times with ethanol to obtain a 20 mmol / mL flower-shaped ZIF-67 solution. Step 4: Electrodeposition of g-C3N4 film First, a copper foil with a thickness of 1 mm was cut into a size of 5 mm × 10 mm and cleaned with acetone, ethanol and deionized water for later use; After 5 mg of PEI-functionalized g-C3N4 nanosheets prepared in step 2 were ultrasonically dispersed in 30 mL of ethanol for 2 h, 3 mg of AlCl3·6H2O was added to promote the deposition of g-C3N4 nanosheets to obtain a g-C3N4 nanosheet suspension; The cleaned copper foil and platinum sheet of the same size were placed in the g-C3N4 nanosheet suspension as electrodes for the electrodeposition experiment, respectively. The distance between the two electrodes was controlled to be 4 cm, the deposition time was 240 seconds, and the deposition voltage was 24 V. The positively charged g-C3N4 nanosheet moved toward the copper foil electrode and finally deposited on its surface. The copper foil was thoroughly rinsed with ethanol three times and then dried in a vacuum oven at 45 ° C for 1 hour to obtain g-C3N4 nanosheet / copper foil. Step 5, preparing g-C3N4 / ZIF-67 composite surface enhanced Raman scattering substrate by secondary electrodeposition The g-C3N4 / ZIF-67 composite substrate is prepared by modifying ZIF-67 on g-C3N4 nanosheets / copper foil by secondary electrodeposition process. Using a two-electrode system, the g-C3N4 nanosheets / copper foil prepared in step 4 is used as the negative electrode, and the platinum sheet is used as the positive electrode, and is placed in a flower-shaped ZIF-67 solution with a concentration of 20mmol / mL. By adjusting the deposition voltage to 12V within a constant time of 300 seconds, flower-shaped ZIF-67 with different densities is grown on the surface of the g-C3N4 nanosheets / copper foil, and the g-C3N4 / ZIF-67 composite surface enhanced Raman scattering substrate is obtained by washing and drying.

[0025] Figure 5 Show the scanning electron microscope photographs of the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared in this example. From Figure 5 it can be seen that the g-C3N4 nanosheets are densely coated on the copper foil, and ZIF-67 microflowers are scattered on it, and the two form a good composite.

[0026] Drop 1.0×10 -4 M Aβ42 standard solution on the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate and incubate for 6 hours. Finally, immediately measure the sample using a Raman microscope. The excitation wavelength of the Raman spectrometer is 532 nm, the laser power is 1 mW, and the acquisition time is 10 s. Figure 6 Is the SERS spectrum of Aβ42 detected using the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared in this example. From Figure 6 it can be seen that the intensity of Aβ42 at 1365 wavenumbers is 843.

[0027] Sensitivity detection test of the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared in Example 4 and Example 2.

[0028] Drop a series of Aβ42 standard solutions with different concentrations on the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared in Example 2 and incubate for 6 hours. Finally, immediately measure the sample using a Raman microscope. The excitation wavelength of the Raman spectrometer is 532 nm, the laser power is 1 mW, and the acquisition time is 10 s.

[0029] Figure 7 Is the SERS spectrum of Aβ42 detected using the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared in Example 2. The concentration of Aβ42 varies from 1×10 -8 to 1×10 -4 M. From Figure 7 it can be seen that as the concentration of Aβ42 increases, the SERS signal intensity also gradually increases.

[0030] Figure 8 Is the corresponding dose-response curve of the peak intensity at 1365 cm -1 . From Figure 8 it can be seen that in the range of 1×10 -8 to 1×10 -4 M, the SERS signal intensity shows a good linear relationship with the logarithm of the Aβ42 concentration. The linear regression equation is y = 188.82x + 1530.57, R 2= 0.986. Based on three times the signal-to-noise ratio, the detection limit was estimated to be 8.1×10 -9 M.

[0031] Example 5, Comparative Experiment.

[0032] Control Group: A g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate was prepared by physical mixing as follows: g-C3N4 nanosheets / copper foil were placed in a flower-like ZIF-67 solution with a concentration of 15 mmol / mL, slowly stirred for 30 minutes, washed and dried to obtain a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate. The preparation methods of the g-C3N4 nanosheets / copper foil and the flower-like ZIF-67 solution were the same as those in Example 2 above. A 1.0×10 -4 M Aβ42 standard solution was dropped onto the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate of the control group and incubated for 6 hours. Finally, the sample was immediately measured using a Raman microscope. The excitation wavelength of the Raman spectrometer was 532 nm, the laser power was 1 mW, and the acquisition time was 10 seconds.

[0033] Figure 9 The SERS spectrum of Aβ42 detected using the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared in this example is shown. As Figure 9 can be seen, the intensity of Aβ42 at 1365 wavenumbers was only 177, which was only about 1 / 5 of that of the composite surface-enhanced Raman scattering substrate prepared by the two-step electrochemical method in Examples 1-3. It can be seen that the composite surface-enhanced Raman scattering substrate prepared by the electrodeposition process in Examples 1-3 of the present invention significantly improved the enhancement degree of the SERS signal.

[0034] The above description is not a limitation of the present invention, nor is the present invention limited to the above examples. Changes, modifications, additions, or substitutions made by those of ordinary skill in the art within the essential scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A preparation method of a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate, characterized in that The following steps are involved: Step 1, ultrasonically dispersing PEI functionalized g-C3N4 nanosheets in ethanol for 0.5-2 hours, and then adding AlCl3·6H2O to obtain a g-C3N4 nanosheet suspension; placing the cleaned copper foil and platinum foil in the g-C3N4 nanosheet suspension, and after electrodeposition, thoroughly rinsing the copper foil with ethanol and vacuum drying to obtain g-C3N4 nanosheets / copper foil, wherein the mixing ratio of PEI functionalized g-C3N4 nanosheets, AlCl3·6H2O and ethanol is: 5 mg: 1-3 mg: 10-30 mL; Step 2: The flower-shaped ZIF-67 is modified on the g-C3N4 nanosheets / copper foil by secondary electrodeposition to prepare a g-C3N4 / ZIF-67 composite surface enhanced Raman scattering substrate.

2. The preparation method of a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate according to claim 1, wherein The synthesis of PEI functionalized g-C3N4 nanosheets described in step 1 comprises the following steps: Step 1-1, preparation of g-C3N4 nanosheets: dissolve bulk g-C3N4 in concentrated hydrochloric acid, stir evenly to obtain a g-C3N4 suspension with a concentration of 15-20 mM, perform ultrasonic treatment until its color changes from light green to milky white, transfer the milky white suspension to a polytetrafluoroethylene container, react at 70-90 ° C for 12-24 hours, centrifuge to obtain the supernatant, wash the precipitate with deionized water and then centrifuge to obtain the supernatant, after multiple centrifugation and washing, collect the supernatant obtained from each centrifugation to obtain the g-C3N4 supernatant; Step 1-2, adding 20-30 mg / mL polyethyleneimine aqueous solution dropwise to the g-C3N4 supernatant obtained in step 1, and after fully mixing under ultrasonic treatment, the mixture is continuously stirred at a rate of 120-180rpm for 12-24 hours, centrifuged to obtain the supernatant, the precipitate is washed with deionized water and then centrifuged to obtain the supernatant, after multiple centrifugation and washing, the supernatant obtained from each centrifugation is collected and vacuum dried to obtain PEI functionalized g-C3N4 nanosheets, wherein the mass ratio of polyethyleneimine aqueous solution to g-C3N4 supernatant is controlled to be 2:

1.

3. The preparation method of a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate according to claim 1, characterized in that In step 1, the distance between the two electrodes during the electrodeposition process is 2-4 cm, the deposition time is controlled to be 60-240 seconds, and the deposition voltage is 15-24V.

4. The preparation method of a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate according to claim 1, characterized in that Step 2 is as follows: using a two-electrode system, the g-C3N4 nanosheet / copper foil prepared in step 1 is used as the negative electrode and the platinum sheet is used as the positive electrode, and is placed in a flower-shaped ZIF-67 solution with a concentration of 10-20 mmol / mL, and the deposition voltage is adjusted to 6-12 V within a constant time of 300 seconds, and flower-shaped ZIF-67 with different densities is grown on the surface of the g-C3N4 nanosheet / copper foil, which is washed and dried to obtain a g-C3N4 / ZIF-67 composite surface enhanced Raman scattering substrate.

5. The preparation method of a g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate according to claim 4, characterized in that The preparation of the flower-like ZIF-67 solution comprises the following steps: preparing a 2-methylimidazole solution with a concentration of 14-140 mg / mL and a Co(NO3)2·6H2O solution with a concentration of 21-210 mg / mL respectively using methanol. After fully mixing the 2-methylimidazole solution and the Co(NO3)2·6H2O solution at a volume ratio of 1:3, incubating for 1-3 hours at room temperature, and then washing with ethanol several times, a flower-like ZIF-67 solution with a concentration of 10-20 mmol / mL is obtained.

6. Use of the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared by the method according to any one of claims 1-5 in the detection of Aβ protein. This method is not for the purpose of diagnosis or treatment, and is characterized in that: The Aβ protein solution to be measured is modified on the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate. After incubating for 4-8 hours, the sample is immediately measured using a Raman microscope. According to the linear relationship between the Raman intensity and the concentration of the Aβ protein solution, the concentration of the Aβ protein in the Aβ protein solution to be measured is calculated.

7. Use of the g-C3N4 / ZIF-67 composite surface-enhanced Raman scattering substrate prepared by the method according to any one of claims 1-5 in the preparation of a diagnostic reagent for Alzheimer's disease.

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