Preparation method of VP@UiO-66-NH2 / Ag NPs composite SERS chip and application thereof in detection of A beta protein

Abstract

The application discloses a preparation method of a VP@UiO-66-NH2 / Ag NPs composite SERS chip and application thereof in A beta protein detection, and is characterized by comprising the following steps: synthesizing a VP-graphite paper base by a chemical vapor transport method on a surface of a graphite paper to obtain a VP-graphite paper base; synthesizing a UiO-66-NH2 layer on the surface of the VP-graphite paper base by a solvothermal method to obtain a VP@UiO-66-NH2 composite base; and finally, placing the composite base in an ion sputtering instrument, uniformly sputtering silver nanoparticles on the surface of the composite base, taking out the composite base, and obtaining the VP@UiO-66-NH2 / Ag NPs composite SERS chip, wherein the VP@UiO-66-NH2 / Ag NPs composite SERS chip has the advantages of high sensitivity, good stability and uniformity, controllable preparation process, low cost, application in Alzheimer's disease biomarker detection, high specificity in identification and quantitative analysis of A beta 40 and A beta 42 proteins, fast response and good stability.

Description

Technical Field

[0001] This invention belongs to the field of materials science and analytical chemistry, specifically relating to the preparation method of VP@UiO-66-NH2 / Ag NPs composite SERS chip and its application in Aβ protein detection. Background Technology

[0002] With the high incidence of Alzheimer's Disease (AD) in the elderly population and its profound impact on social health, early diagnosis and research into its pathological mechanisms have become major challenges in neuroscience. Abnormal aggregation of β-amyloid protein (Aβ) is one of the core pathological features of AD, with Aβ40 and Aβ42 being the main subtypes. Changes in their levels in brain tissue and body fluids are closely related to disease progression. Compared to Aβ40, Aβ42 has stronger hydrophobicity and a greater tendency to aggregate, making it more prone to forming neurotoxic oligomers and amyloid plaques. Therefore, achieving highly sensitive and specific detection of Aβ40 and Aβ42 not only contributes to a deeper understanding of the pathogenesis of AD but also provides crucial evidence for early screening, disease monitoring, and efficacy evaluation.

[0003] Aβ protein is a biomarker for Alzheimer's disease. Traditional methods for Aβ protein detection include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and mass spectrometry (MS). While ELISA is simple to operate and has high throughput, it is susceptible to antibody cross-reactivity, making it difficult to distinguish highly similar Aβ subtypes, and its sensitivity is limited. Although HPLC and MS offer high accuracy and specificity, they rely on complex sample pretreatment, expensive equipment, and require specialized operation, making rapid, real-time detection difficult. These methods have certain limitations in large-scale clinical screening or dynamic monitoring applications.

[0004] Surface-enhanced Raman spectroscopy (SERS) technology, with its advantages of single-molecule-level sensitivity, fingerprint recognition capability, rapid non-destructive detection, and strong resistance to water interference, has shown significant potential in protein conformation analysis and biomarker detection. Studies have shown that the performance of SERS is highly dependent on the enhancement effect, uniformity, and stability of the substrate material. Therefore, developing SERS substrates with high enhancement factors, good reproducibility, and biocompatibility is crucial for achieving accurate detection of Aβ protein. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing a VP@UiO-66-NH2 / Ag NPs composite SERS chip with high sensitivity, good stability and uniformity, controllable preparation process and low cost, and its application in Aβ protein detection. When applied to the detection of biomarkers for Alzheimer's disease, it can achieve highly specific identification and quantitative analysis of Aβ40 and Aβ42 proteins, and has a fast response and good stability.

[0006] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: a method for preparing a VP@UiO-66-NH2 / Ag NPs composite SERS chip, comprising the following steps: Step 1: Using red phosphorus as a precursor, purple phosphorus is synthesized on the surface of graphite paper by chemical vapor transport method to obtain VP-graphite paper substrate; Step 2: The VP-graphite paper substrate obtained in Step 1 is subjected to a solvothermal method to synthesize a UiO-66-NH2 layer, thereby obtaining a VP@UiO-66-NH2 composite substrate; Step 3: Place the VP@UiO-66-NH2 composite substrate obtained in Step 2 in an ion sputtering instrument, uniformly sputter silver nanoparticles onto its surface and remove it to obtain the VP@UiO-66-NH2 / Ag NPs composite SERS chip.

[0007] Further, step 1 is as follows: After cutting the graphite paper into small pieces, it is ultrasonically cleaned in acetone, ethanol and deionized water in sequence, then irradiated with ultraviolet light to remove organic residues on the surface and dried. Then, a thin tin film is deposited on its surface by thermal evaporation. The graphite paper coated with red phosphorus powder, iodine and Sn is vacuum sealed in a quartz ampoule. The sealed quartz tube is placed in a tube furnace and heated to 600-650 °C at 2-10 °C / min to fully vaporize the red phosphorus (RP). Then, it is slowly cooled to 450-500 °C at 0.5-2 °C / min to obtain graphite paper modified with purple phosphorus, i.e., VP-graphite paper substrate.

[0008] Further, step 2 is as follows: the VP-graphite paper substrate obtained in step 1 is cut into square pieces and immersed in zirconium tetrachloride solution for 10-15 hours; Zr 4+ The functionalized VP-graphite paper substrate was transferred to an NH2-BDC ligand solution, and glacial acetic acid was added. After stirring at room temperature, a mixed system was obtained. The mixed system was transferred to a high-pressure reactor and subjected to a solvothermal reaction at 100-150℃ for 10-15 h. After the system cooled naturally to room temperature, the resulting dark purple VP-graphite paper composite matrix was taken out, and the residue was removed by washing with DMF and methanol in sequence. Then, it was vacuum dried to obtain the VP@UiO-66-NH2 composite substrate.

[0009] Furthermore, both the zirconium tetrachloride solution and the NH2-BDC ligand solution use N,N-dimethylformamide as the solvent, and the molar ratio of zirconium tetrachloride to the NH2-BDC ligand is 1:(0.5-2).

[0010] Furthermore, the amount of glacial acetic acid added in the solvothermal reaction is 6-8 mL to regulate crystal growth kinetics.

[0011] Further, step 3 specifically involves placing the VP@UiO-66-NH2 composite substrate obtained in step 2 into an ion sputtering instrument, using a high-purity silver target, controlling the sputtering current to be 30-40 mA, and the sputtering time to be 40-160 s. After sputtering is completed, the VP@UiO-66-NH2 / Ag NPs composite SERS chip is obtained.

[0012] This invention also provides the application of the above-mentioned VP@UiO-66-NH2 / Ag NPs composite SERS substrate in the detection of Aβ protein. This application is not for disease diagnosis. The above-mentioned SERS substrate is co-incubated with 10-15 µL of a test solution containing Aβ40 and / or Aβ42 protein. After washing and drying, detection is performed by Raman spectroscopy, based on 1585 cm⁻¹. -1 Linear relationship between Raman intensity and Aβ40 solution concentration and / or based on 1656 cm⁻¹ -1 The linear relationship between Raman intensity and Aβ42 solution concentration was used to calculate the concentration of Aβ40 and / or Aβ42 protein in the test solution.

[0013] Compared with existing technologies, the advantages of this invention are as follows: This invention provides a method for preparing a VP@UiO-66-NH2 / Ag NPs composite SERS chip and its application in Aβ protein detection. It is the first to propose combining a purple phosphorus (VP) array with an aminated metal-organic framework material (UiO-66-NH2) and loading silver nanoparticles (Ag NPs) for SERS chip preparation. This VP@UiO-66-NH2 / Ag NPs composite substrate combines the unique layered structure and excellent charge transfer capability of the VP array, the high specific surface area and specific adsorption enrichment of UiO-66-NH2, and the strong localized surface plasmon resonance effect of Ag NPs. These three factors synergistically and significantly enhance the intensity, uniformity, and stability of the SERS signal. Compared with other low-dimensional material-MOF composite systems, the composite material of this invention has significant advantages in structural design, enhancement mechanism, and biomolecular affinity.

[0014] The preparation method of this invention involves in-situ growth of a VP array via hydrothermal method and precise loading of AgNPs using ion sputtering technology. This process is controllable, low-cost, and exhibits good reproducibility and potential for large-scale production. By precisely controlling the hydrothermal parameters and sputtering conditions, the uniformity, consistency, and high density of active "hot spots" in the composite substrate nanostructure are ensured, resulting in excellent reliability and stability in practical bioassay applications.

[0015] The SERS substrate of this invention enables highly sensitive and specific detection of Aβ40 and Aβ42 proteins, key biomarkers of Alzheimer's disease, with detection limits reaching 10⁻⁶ for both. -10 The assay exhibits extremely high detection sensitivity at the mg / mL level. Thanks to the selective enrichment of proteins by UiO-66-NH2 and the synergistic enhancement effect of VP and Ag NPs, selective capture and signal amplification of Aβ40 and Aβ42 are achieved. This substrate demonstrated good signal reproducibility in multiple assays, providing a new technical approach for the ultrasensitive detection and real-time analysis of early Alzheimer's disease biomarkers.

[0016] The SERS substrate of this invention is not only suitable for the detection of Aβ40 and Aβ42 proteins, but its functionalized surface can also be modified with different aptamers or antibodies to further detect other neurodegenerative disease-related proteins (such as Tau protein, α-synuclein, etc.), various inflammatory factors, or nucleic acid markers. The detection limits can reach the picomolar or even femtomolar level, meeting the high-sensitivity, multi-target detection needs of trace biomarkers in clinical body fluids or tissue samples, and has broad application prospects in early disease diagnosis and dynamic monitoring.

[0017] In summary, the present invention provides a method for preparing a VP@UiO-66-NH2 / Ag NPs composite SERS chip and its application in Aβ protein detection. This chip possesses a three-dimensional hierarchical structure composed of a purple phosphorus (VP) array, a UiO-66-NH2 porous layer, and surface Ag nanoparticles. This structure exhibits highly ordered pores and uniformly distributed SERS "hot spots," resulting in extremely high sensitivity. Furthermore, the Raman signal enhancement factor of its SERS substrate is ≥10. 8 With a relative standard deviation (RSD) ≤ 8.0%, it exhibits good signal reproducibility and spatial homogeneity, making it suitable for the quantitative analysis of trace biomolecules. Attached Figure Description

[0018] Figure 1 A scanning electron microscope (SEM) image of the VP array prepared in Example 1 of the present invention; Figure 2 Scanning electron microscope (SEM) image of the VP@UiO-66-NH2 composite substrate prepared in Example 1 of this invention; Figure 3 Scanning electron microscope (SEM) image of the VP@UiO-66-NH2 / Ag NPs composite SERS chip prepared for Example 1 of the present invention; Figure 4 The image shows a scanning electron microscope (SEM) image of the VP@UiO-66-NH2 / Ag NPs composite prepared in Example 2. Figure 5 The image shows a scanning electron microscope (SEM) image of the VP@UiO-66-NH2 / Ag NPs composite prepared in Example 3. Figure 6 This is a scanning electron microscope (SEM) image of the VP@UiO-66-NH2 / Ag NPs composite prepared in Example 4; Figure 7 In step 2 of Example 1, at 10 -7 Raman spectra of VP@UiO-66-NH2SERS chips with different ratios of metal ions and organic ligands in UiO-66-NH2 under M MB; Figure 8 In step 3 of Example 1, at 10 -7 Raman spectra of VP@UiO-66-NH2 / Ag NPs SERS chips recombined with Ag NPs at different sputtering times under M MB; Figure 9 The sample prepared in Example 1 at 10 -7 Raman spectra of VP, VP@UiO-66-NH2, and VP@UiO-66-NH2 / Ag NPsSERS chips under M MB; Figure 10 In Example 1, at 10 -7 Figure showing the repeatability test results of the VP@UiO-66-NH2 / Ag NPs composite SERS chip under M MB; Figure 11 In Example 1, at 10 -7 Stability test results of VP@UiO-66-NH2 / Ag NPs composite SERS chip under M MB; Figure 12 In Example 1, at 10 -7 The detection limit test results of the VP@UiO-66-NH2 / Ag NPs composite SERS chip under M MB; Figure 13 Raman spectra of Aβ40 protein molecule solutions of different concentrations dropped onto the VP@UiO-66-NH2 / Ag NPs composite SERS chip prepared in Example 1; Figure 14Raman spectra of Aβ42 protein molecule solutions of different concentrations dropped onto the VP@UiO-66-NH2 / Ag NPs composite SERS chip prepared in Example 1; Figure 15 Raman spectra of Aβ40 and Aβ42 mixed in different ratios (Aβ40:Aβ42=20:1, 10:1, 1:1) in different body fluids, where CSF refers to cerebrospinal fluid, Serum refers to serum, and Tear refers to tears. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0020] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available products. The BWS415 Raman spectrometer used in the examples was purchased from B&W Tek Inc., USA. The purity of red phosphorus was ≥99.99%, the purity of zirconium tetrachloride was ≥99.9%, the purity of 2-aminoterephthalic acid (NH2-BDC) was ≥98.0%, and the purity of the silver target was ≥99.99%. I. Specific Implementation Methods Example 1: A method for fabricating a composite SERS chip based on VP@UiO-66-NH2 / Ag NPs, comprising the following steps: Step 1: Fabrication of Purple Phosphorus VP Array VP crystals were synthesized via chemical vapor transport (CVT): red phosphorus (RP) was used as a precursor and thoroughly ground in an agate mortar; graphite paper was cut into approximately 5 × 10 mm fragments and then ultrasonically cleaned in acetone, ethanol, and deionized water for 15 min each, followed by ultraviolet (UV) irradiation for 20 min to remove surface organic residues; subsequently, the graphite substrate was dried in a vacuum drying oven at 60 °C for 12 h, and a thin tin (Sn) film was deposited on its surface using a thermal evaporation method; the treated RP, iodine (I2, transport agent), and Sn-coated graphite paper were then vacuum-sealed into a quartz ampoule with a diameter of 1 cm (vacuum degree <10). -2 Pa); The sealed quartz tube is placed in a tube furnace, and the temperature is first raised to 620 ℃ at 5 ℃ / min to fully vaporize RP, and then slowly cooled to 485 ℃ at 1 ℃ / min to promote the crystallization and precipitation of VP on the Sn-coated graphite substrate, thus obtaining graphite paper modified with VP surface, abbreviated as VP-graphite paper substrate; Step 2: Preparation of VP@UiO-66-NH2 composite substrate First, 102 mg of zirconium tetrachloride was dissolved in 25 mL of N,N-dimethylformamide (DMF) and sonicated at 25 °C (40 kHz, 120 W) for 10 min to obtain a zirconium tetrachloride solution. Then, 72.5 mg of 2-aminoterephthalic acid (NH2-BDC) was dissolved in 25 mL of N,N-dimethylformamide (DMF) and sonicated at 25 °C (40 kHz, 120 W) for 10 min to obtain an NH2-BDC ligand solution. VP-graphite paper substrates were cut into 5 mm × 5 mm square pieces and immersed in the zirconium tetrachloride solution. The mixture was then allowed to stand at room temperature for 12 h in a closed system to achieve Zr... 4+ Sufficient coordination and pre-adsorption on the surface of graphite paper; the above Zr 4+ The functionalized VP-graphite paper substrate was transferred to an NH2-BDC ligand solution, and 6 mL of glacial acetic acid (HOAc) was added to adjust the crystal growth kinetics. After stirring at room temperature for 20 min, the mixture was transferred to a stainless steel high-pressure reactor lined with polytetrafluoroethylene and reacted solvothermically at 120 °C for 12 h. After the system cooled naturally to room temperature, the resulting deep purple VP-graphite paper composite matrix was taken out and washed three times each with DMF (3×8 mL) and methanol (3×8 mL) to remove residues. Finally, it was vacuum dried at 60 °C for 12 h to obtain the VP@UiO-66-NH2 composite substrate. Step 3: Fabrication of VP@UiO-66-NH2 / Ag NPs composite chip The VP@UiO-66-NH2 composite substrate obtained in step 2 was placed in an ion sputtering instrument, and silver nanoparticles were uniformly sputtered onto its surface for 120 seconds before being removed, thus obtaining the VP@UiO-66-NH2 / Ag NPs composite SERS chip.

[0022] Figure 1 This is a scanning electron microscope (SEM) image of the VP prepared in this embodiment. Figure 1 As shown, the prepared VP array consists of a large number of uniform micro-plates interleaved and inserted to form a network trap structure with a large number of active surfaces and edges.

[0023] Figure 2 This is a scanning electron microscope (SEM) image of the VP@UiO-66-NH2 composite prepared in this embodiment. Figure 2As shown, a network trap structure with numerous active surfaces and edges is formed in the prepared VP array, which consists of a large number of uniformly interleaved microplates. During the in-situ growth of UiO-66-NH2, particles are uniformly distributed on the exposed surfaces and edges of the VP, and 100-200 nm octahedral UiO-66-NH2 crystals are uniformly anchored on the surface of the sheets, providing a high density of -NH2 active sites.

[0024] Figure 3 This is a scanning electron microscope (SEM) image of the VP@UiO-66-NH2 / Ag NPs composite prepared in this embodiment. Figure 3 As shown, this composite exhibits unique microstructural features. Scanning electron microscopy (SEM) images of the VP@UiO-66-NH2 / AgNPs composite SERS substrate prepared in this embodiment reveal a three-tiered structure: a VP two-dimensional sheet array, UiO-66-NH2 octahedrons, and Ag nanoparticles. The bottom layer consists of flexible purple phosphorus sheets, 2–5 μm in lateral width, overlapping to form a continuous conductive network. 100–200 nm octahedral UiO-66-NH2 crystals are uniformly anchored on the sheet surface, with exposed crystal faces providing high-density -NH2 active sites. Subsequently, 10–30 nm Ag NPs are selectively deposited on the edges and vertices of the octahedrons, forming a high-density "hot-spot" network in three-dimensional space. This configuration organically integrates the rapid electron transport of the VP sheets, the high specific surface area and molecular enrichment capacity of UiO-66-NH2, and the local electromagnetic enhancement effect of Ag NPs, achieving morphology-interface synergy and laying the structural foundation for high-sensitivity and high-stability SERS detection.

[0025] Example 2 is the same as Example 1 above, except that in step 3, the VP@UiO-66-NH2 composite substrate obtained in step 2 is placed in an ion sputtering instrument, and silver nanoparticles are uniformly sputtered on its surface for 40 seconds before being taken out, thus obtaining the VP@UiO-66-NH2 / Ag NPs composite SERS chip.

[0026] Figure 4 This is a scanning electron microscope (SEM) image of the VP@UiO-66-NH2 / Ag NPs composite prepared in this embodiment. Figure 4 As shown, this composite exhibits unique microstructural features. Nanoscale noble metal particles are selectively deposited on the surface of VP@UiO-66-NH2, forming a dense signal enhancement network within the space. This structure achieves efficient synergy between morphology and interface by integrating the advantages of each component—charge transport in the bottom layer, molecular enrichment in the middle layer, and electromagnetic enhancement in the surface layer—laying a solid foundation for high-sensitivity and high-stability SERS detection.

[0027] Example 3 is the same as Example 1 above, except that in step 3, the VP@UiO-66-NH2 composite substrate obtained in step 2 is placed in an ion sputtering instrument, and silver nanoparticles are uniformly sputtered on its surface for 80 seconds before being taken out, thus obtaining the VP@UiO-66-NH2 / Ag NPs composite SERS chip.

[0028] Figure 5 This is a scanning electron microscope (SEM) image of the VP@UiO-66-NH2 / Ag NPs composite prepared in Example 3 of this study. Figure 5 As shown, this composite exhibits unique microstructural features. The surface of the VP@UiO-66-NH2 material is selectively modified to form a dense reinforcing network composed of nano-noble metal particles. This structure achieves efficient synergy between multi-level morphology and interfacial properties by orderly integrating the functional advantages of bottom-layer charge transport, middle-layer molecular enrichment, and surface electromagnetic enhancement, thus providing a solid material foundation for high-sensitivity and high-stability SERS detection.

[0029] Example 4 is the same as Example 1 above, except that in step 3, the VP@UiO-66-NH2 composite substrate obtained in step 2 is placed in an ion sputtering instrument, and silver nanoparticles are uniformly sputtered on its surface for 160s before being taken out, thus obtaining the VP@UiO-66-NH2 / Ag NPs composite SERS chip.

[0030] Figure 6 This is a scanning electron microscope (SEM) image of the VP@UiO-66-NH2 / Ag NPs composite prepared in Example 4 of this paper. Figure 6 As shown, this composite exhibits unique microstructural features. A spatially dense reinforcement network was constructed by selectively depositing noble metal nanoparticles on the VP@UiO-66-NH2 surface. This design integrates the advantages of each component: the bottom layer ensures charge transport, the middle layer achieves molecular enrichment, and the surface layer provides electromagnetic enhancement. These three elements synergistically optimize the morphology and interface properties, forming a solid foundation for achieving high-sensitivity and high-stability SERS detection.

[0031] II. Analysis of Experimental Results 1. Optimization of the ratio of metal ions to organic ligands in the VP@UiO-66-NH2 composite substrate The preparation method of the VP@UiO-66-NH2 composite substrate is the same as that in the above specific embodiments. The difference is that the molar ratio of zirconium tetrachloride to 2-aminoterephthalic acid is set to 1:2, 1:1.5, 1:1, and 1:0.5 respectively during the preparation process. Four different VP@UiO-66-NH2 composite substrates were obtained and Raman spectroscopy was performed.

[0032] The results are as follows Figure 7As shown, the SERS activity of UiO-66-NH2 is significantly dependent on the ratio of metal to ligand in its synthesis. When the ligand ratio increases from insufficient (1:0.5) to near the stoichiometric ratio (1:1), the SERS signal is significantly enhanced; however, when the ligand is further in excess (1:2), the signal decreases. This non-monotonic variation can be attributed to the regulatory effect of the ligand ratio on the structure and surface properties of the MOF matrix: at the optimal ratio (1:1), the formed UiO-66-NH2 exhibits high crystallinity, well-developed pores, and a uniform distribution of surface amino functional groups. This provides an ideal platform for the subsequent uniform loading of silver nanoparticles, constructing a dense plasma 'hot spot,' and effectively concentrates the target molecules into the enhanced region through its strong adsorption and enrichment capabilities, thereby achieving synergistic maximization of the SERS signal. When ligands are in excess, the excessive organic ligands may cause pore blockage or surface passivation, which not only hinders the mass transfer and enrichment of analytes but may also interfere with the optimized deposition of silver nanoparticles, leading to a decrease in the number and quality of 'hot spots' and thus a decline in overall SERS performance. Therefore, setting the molar ratio of zirconium tetrachloride to 2-aminoterephthalic acid to 1:1 is the optimal value for achieving MOF structural integrity, adsorption functionality, and surface modification, and this optimal value was used in subsequent experiments.

[0033] 2. Optimization of silver particle sputtering time Based on the studies in Examples 1, 2, 3, and 4, it was found that Ag sputtering time is another key parameter for controlling the SERS performance of the VP@UiO-66-NH2@Ag NPs porous array. Figure 8 As shown, the SERS signal intensity exhibits a non-monotonic trend of first increasing and then decreasing with increasing sputtering time. This pattern profoundly reveals the structure-property relationship between the morphology of noble metal nanostructures and the plasmon resonance enhancement effect: at shorter sputtering times, the formed Ag nanoparticles are sparse and small, unable to construct sufficient electromagnetic field coupling 'hot spots,' resulting in limited overall enhancement capability. At the optimal sputtering time of 120 seconds, Ag nanoparticles grow to a suitable size and are uniformly distributed at a moderate density, forming a large number of high-intensity, high-density 'hot spot' networks between the particles. This structure also achieves resonance matching with the excitation wavelength, thereby converting the excellent molecular enrichment capability of the MOF substrate into a very strong SERS response, maximizing the signal intensity. When the sputtering time is too long (160 s), excessive Ag deposition leads to excessive growth and aggregation of nanoparticles, tending to form a continuous film, causing the quenching of key nano-interstitial 'hot spots' and deteriorating the plasmon resonance characteristics. At the same time, an excessively thick Ag layer will cover and block the diffusion paths of the MOF channels. Therefore, the SERS performance decreases significantly.

[0034] 3. Analysis of the synergistic effect of Raman intensity on composite SERS chips based on VP@UiO-66-NH2 / Ag NPs In a specific embodiment, a multi-level composite SERS substrate of VP@UiO-66-NH2@Ag NPs was rationally constructed using a three-step method. For example... Figure 9 As shown, the necessity of the synergistic design of the functional components is confirmed: First, the three-dimensional porous VP framework provides mechanical support and conductive channels for the entire system, contributing a fundamental chemical enhancement effect. Then, the introduced UiO-66-NH2 layer, with its ultra-high specific surface area and functional surface, acts as a highly efficient 'molecular enricher,' concentrating trace target molecules into the detection region, solving the core problem of low molecular detection rate at low concentrations, and resulting in a first leap in signal intensity. Finally, the modified Ag nanoparticles construct a plasmonic 'hotspot' network on the enrichment layer surface, providing extremely strong electromagnetic field enhancement. This design allows the molecules enriched by the MOF to be located maximally in the region of strongest electromagnetic field enhancement, thereby converting the enrichment effect into extremely high signal output and achieving a significant improvement in SERS sensitivity.

[0035] In summary, the VP@UiO-66-NH2@Ag NPs multi-level composite SERS substrate achieves a synergistic effect of electromagnetic enhancement, chemical enhancement, and molecular enrichment at the nanoscale, resulting in a 1+1 greater than 2 effect and significantly improving SERS intensity and detection sensitivity.

[0036] 4. Repeatability and stability analysis of composite SERS chip based on VP@UiO-66-NH2 / Ag NPs Raman surface scanning was performed on the VP@UiO-66-NH2 / Ag NPs composite SERS chip prepared in Example 1, and the characteristic peak intensity of methylene blue (MB) at 1600 measurement points was analyzed. The results are as follows: Figure 10 As shown, the relative standard deviation (RSD) of its signal intensity is less than 8%. This is due to the uniform growth of MOF guided by VP and the consistent deposition of Ag NPs, which forms a highly uniform "hot spot" distribution on a macroscopic scale, ensuring high reproducibility and spatial reliability of the detection results, and resulting in excellent signal uniformity.

[0037] like Figure 11 As shown, after storing the composite SERS chip based on VP@UiO-66-NH2 / Ag NPs prepared in Example 1 at room temperature for 10 weeks, its SERS characteristic peak for MB (1621 cm⁻¹) was observed. -1 The strength retention rate remains above 86%, indicating structural stability and suitability for practical applications. This is attributed to the robust anchoring of Ag NPs by the MOF rigid framework, which effectively prevents oxidation and aggregation, ensuring long-term reliability and giving the SERS chip excellent stability.

[0038] 5. Sensitivity analysis of the composite SERS chip of VP@UiO-66-NH2 / Ag NPs MB was used as a model molecule to assess the general sensitivity of the substrate. Results are as follows: Figure 12 As mentioned above, in 10 -10 M to 10 -5 Within the M concentration range, the characteristic peak intensity of MB exhibits a linear relationship with the logarithm of concentration, with a linear correlation coefficient R² > 0.99. The results indicate that this chip can detect concentrations as low as 10 M. -10 The high concentration of MB molecules in M ​​demonstrates high detection sensitivity, providing strong support for trace analysis.

[0039] III. Application Cases 1. Detection and analysis of Aβ40 and Aβ42 based on a composite SERS chip of VP@UiO-66-NH2 / Ag NPs.

[0040] Figure 13 This is the Raman spectrum of the Aβ40 protein in the VP@UiO-66-NH2 / Ag NPs composite SERS chip prepared in Example 1. From... Figure 13 As can be seen from this, with the increase of Aβ40 molecule concentration, 1585 cm⁻¹ -1 The intensity of the Raman characteristic peaks also increased accordingly, indicating that the SERS chip has good detection capability for Aβ40 protein molecules. Furthermore, the clear characteristic peaks in the Raman spectrum also demonstrate that the chip has a high Raman signal enhancement effect, which can effectively improve detection sensitivity.

[0041] Figure 14 This is the Raman spectrum of the Aβ42 protein in the VP@UiO-66-NH2 / Ag NPs composite SERS chip prepared in Example 1. From... Figure 14 As can be seen from this, with the increase of Aβ42 protein molecule concentration, 1656 cm -1 The intensity of the Raman characteristic peaks also increased accordingly, indicating that the SERS chip has good detection capability for Aβ42 protein molecules. Furthermore, the clear characteristic peaks in the Raman spectrum also demonstrate that the chip has a high Raman signal enhancement effect, which can effectively improve detection sensitivity.

[0042] 2. Raman spectra of Aβ40 and Aβ42 were measured in different body fluids (cerebrospinal fluid, serum, and tears) after mixing them in different ratios (Aβ40:Aβ42=20:1, 10:1, 1:1).

[0043] Figure 15To test the Raman spectra of Aβ40 and Aβ42, Aβ40 and Aβ42 were mixed in different ratios (Aβ40:Aβ42 = 20:1, 10:1, 1:1) and added to different body fluids (cerebrospinal fluid CSF, serum, and tear fluid). Then, 10-15 μL of the sample solution was added to the composite SERS chip based on VP@UiO-66-NH2 / Ag NPs prepared in Example 1, and incubated for 8 hours. Raman characteristic peaks of Aβ40 and Aβ42 were detected in different samples at different mixed concentrations. As the concentration of Aβ42 in the Aβ40:Aβ42 concentration ratio decreased, the detected characteristic peak of Aβ42 gradually weakened.

[0044] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A method for fabricating a VP@UiO-66-NH2 / Ag NPs composite SERS chip, characterized in that... Includes the following steps: Step 1: Using red phosphorus as a precursor, purple phosphorus is synthesized on the surface of graphite paper by chemical vapor transport method to obtain VP-graphite paper substrate; Step 2: The VP-graphite paper substrate obtained in Step 1 is subjected to a solvothermal method to synthesize a UiO-66-NH2 layer, thereby obtaining a VP@UiO-66-NH2 composite substrate; Step 3: Place the VP@UiO-66-NH2 composite substrate obtained in Step 2 in an ion sputtering instrument, uniformly sputter silver nanoparticles onto its surface and remove it to obtain the VP@UiO-66-NH2 / Ag NPs composite SERS chip.

2. The method for preparing a VP@UiO-66-NH2 / Ag NPs composite SERS chip according to claim 1, characterized in that... Step 1 is as follows: After cutting the graphite paper into small pieces, it is ultrasonically cleaned in acetone, ethanol and deionized water in sequence. Then, it is dried after removing the organic residue on the surface by ultraviolet irradiation. A thin tin film is then deposited on its surface by thermal evaporation. The graphite paper coated with red phosphorus powder, iodine and Sn is vacuum sealed in a quartz ampoule. The sealed quartz tube is placed in a tube furnace and heated to 600-650 °C at 2-10 °C / min to fully vaporize the red phosphorus. Then, it is slowly cooled to 450-500 °C at 0.5-2 °C / min to obtain graphite paper modified with purple phosphorus, i.e., VP-graphite paper substrate.

3. The method for fabricating a VP@UiO-66-NH2 / Ag NPs composite SERS chip according to claim 1, characterized in that... Step 2 is as follows: Cut the VP-graphite paper substrate obtained in Step 1 into square pieces and immerse them in zirconium tetrachloride solution for 10-15 hours; then... 4+ The functionalized VP-graphite paper substrate was transferred to an NH2-BDC ligand solution, and glacial acetic acid was added. After stirring at room temperature, a mixed system was obtained. The mixed system was transferred to a high-pressure reactor and subjected to a solvothermal reaction at 100-150℃ for 10-15 h. After the system cooled naturally to room temperature, the resulting dark purple VP-graphite paper composite matrix was taken out, and the residue was removed by washing with DMF and methanol in sequence. Then, it was vacuum dried to obtain the VP@UiO-66-NH2 composite substrate.

4. The method for preparing a VP@UiO-66-NH2 / Ag NPs composite SERS chip according to claim 3, characterized in that: The solvent used in both the zirconium tetrachloride solution and the NH2-BDC ligand solution is N,N-dimethylformamide, and the molar ratio of zirconium tetrachloride to NH2-BDC ligand is 1:(0.5-2).

5. The method for preparing a VP@UiO-66-NH2 / Ag NPs composite SERS chip according to claim 3, characterized in that: The amount of glacial acetic acid added in the solvothermal reaction is 6-8 mL.

6. The method for fabricating a VP@UiO-66-NH2 / Ag NPs composite SERS chip according to claim 1, characterized in that... Step 3 is as follows: Place the VP@UiO-66-NH2 composite substrate obtained in step 2 into an ion sputtering instrument, use a high-purity silver target, control the sputtering current to be 30-40 mA, and the sputtering time to be 40-160 s. After sputtering is completed, the VP@UiO-66-NH2 / AgNPs composite SERS chip is obtained.

7. An application of a VP@UiO-66-NH2 / Ag NPs composite SERS substrate in the detection of Aβ protein, wherein the application is not for disease diagnosis, characterized in that: The SERS substrate according to any one of claims 1-6 was co-incubated with 10-15 µL of the test solution containing Aβ40 and / or Aβ42 protein. After washing and drying, the sample was detected by Raman spectroscopy, based on a 1585 cm⁻¹ value. -1 Linear relationship between Raman intensity and Aβ40 solution concentration and / or based on 1656 cm⁻¹ -1 The linear relationship between Raman intensity and Aβ42 solution concentration was used to calculate the concentration of Aβ40 and / or Aβ42 protein in the test solution.

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