A method for preparing a reusable sers substrate
Patent Information
- Application Number
- CN202610456268.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-08
- Publication Date
- 2026-06-09
Smart Images
Figure CN122171519A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of surface-enhanced Raman scattering (SERS) detection technology, and relates to the preparation of AAO-based SERS substrates, and particularly to a method for preparing reusable SERS substrates. Background Technology
[0002] Surface-enhanced Raman scattering (SERS) refers to the phenomenon where the Raman scattering signal of a analyte is amplified when it is adsorbed onto a rough metal surface. This technique, developed based on Raman spectroscopy, is an emerging surface detection technology that has now reached the level of single-molecule detection. Metal nanostructures containing nanoscale gaps, due to their ability to excite enhanced electromagnetic fields through localized surface plasmon resonance (LSPR), have been studied for achieving ultrasensitive detection and even single-molecule detection. In particular, when molecules are located within nanoscale gaps, the detection sensitivity can be improved by several orders of magnitude compared to inherent weak spontaneous Raman scattering.
[0003] Assembling plasmonic nanostructures with high density and uniformly distributed hot spots from silver nanoparticles (AgNPs) has become crucial for achieving highly sensitive Raman detection. Therefore, extensive research has been conducted in recent years on the preparation of AgNP-based SERS substrates; however, the signal uniformity and batch repeatability need improvement, which limits the commercialization of this substrate.
[0004] Current methods for preparing anodic aluminum oxide (AAO)-based SERS substrates typically involve electrochemically depositing metal within the AAO channels or coating the AAO surface with a metal film via physical vapor deposition (such as evaporation or sputtering). These methods have some drawbacks: electrochemical deposition processes are complex and require high precision in electrolyte and parameter control; the metal films formed by physical vapor deposition are usually continuous films with limited density and uniformity of "hot spots" (regions that enhance the electromagnetic field), and are also costly.
[0005] Therefore, there is an urgent need to develop a simple method for preparing AAO-based SERS substrates and to enable high-sensitivity and high-reproducibility detection of SERS signals. Summary of the Invention
[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing a reusable SERS substrate. The present invention uses an AAO membrane as a substrate and achieves the self-assembly of Ag NPs at the AAO pores through a liquid-liquid interface enrichment method to obtain a SERS substrate, which can be reused in detection.
[0007] This invention is achieved through the following technical solution: A method for preparing a reusable SERS substrate includes the following steps: (1) Preparation of silver nanoparticle sol: Heat a silver nitrate solution of a certain concentration to boiling, quickly add sodium citrate solution, continue the reaction for a certain time, and when the solution turns golden yellow, suspend and magnetically stir until the solution gradually turns gray-green, stop heating, and obtain silver sol; (2) Liquid-liquid separation and purification: The obtained silver sol was centrifuged and washed with deionized water in a centrifuge tube, and then resuspended with deionized water to obtain a silver nanoparticle dispersion. The silver nanoparticle dispersion was poured into an equal volume of n-hexane-ethanol mixed solution, and the solution was allowed to stand for layering. The silver nanoparticles were selectively enriched in the liquid-liquid interface layer. (3) AAO substrate pretreatment: The AAO membrane is first ultrasonically cleaned with ethanol and deionized water, dried with nitrogen, and then treated with oxygen plasma to achieve deep cleaning and superhydrophilization. Finally, the activated membrane is immersed in aminosilane solution for chemical modification. After cleaning and heat treatment, an AAO substrate with amino functional groups covalently bonded to the surface can be obtained. (4) Self-assembly into a film: Take the interfacial suspension from step (2) and drop it evenly onto the surface of the pretreated AAO membrane, and dry it at room temperature to obtain the SERS substrate.
[0008] A further improvement to the present invention is as follows: The concentration of the silver nanoparticle dispersion is 1 mM, and the particle size of the silver nanoparticles is 80~120 nm.
[0009] Furthermore, the volume ratio of hexane to ethanol in the hexane-ethanol mixed solution is 1:0.8~1.2.
[0010] Furthermore, the aminosilane solution is an organic solvent solution of 3-aminopropyltrimethoxysilane (APTMS), and the AAO membrane is immersed in the solution for 1-2 hours.
[0011] Furthermore, the heat treatment process involves baking at 120°C in a vacuum drying oven for 1-2 hours to cure the material.
[0012] Furthermore, in step (4), the interfacial suspension can be added dropwise and dried multiple times to obtain a silver nanoparticle layer of the required thickness.
[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention fundamentally overcomes the bottlenecks of traditional processes, such as particle agglomeration, uneven distribution, weak binding force, and difficulty in forming dense structures, by introducing two core innovations: "liquid-liquid separation and purification" and "capillary-guided self-assembly," on the basis of conventional solution methods. Utilizing the liquid-liquid interface effect of the hexane-ethanol system, well-dispersed silver nanoparticles can be selectively enriched and impurities removed, providing high-quality raw materials for subsequent assembly. Furthermore, leveraging the nanoporous structure of the AAO membrane itself, strong capillary forces are generated during solvent evaporation, actively guiding particles to migrate into the pores and pack tightly, achieving "mechanical interlocking" between the particles and the substrate, forming a robust and dense multilayer nanostructure with a binding strength far exceeding that of ordinary physical adsorption.
[0014] 2. This invention further optimizes SERS performance through meticulous structural design. Specifically, we selected an AAO film with a pore size of approximately 30 nm and a pore spacing of approximately 65 nm, and matched it with silver nanoparticles with a size of approximately 100 nm. This design allows the silver nanoparticles to effectively cover the edges and surfaces of the AAO pores, rather than completely falling into or blocking the pores. This composite structure of "particles on top, pore array below" not only generates strong localized surface plasmon resonance (LSPR) "hot spots" through the gaps between particles, but also may utilize the regular pore array of the AAO as a photonic structure to couple with the LSPR of the silver particles, thereby theoretically achieving a stronger electromagnetic field enhancement effect than a single structure.
[0015] 3. Thanks to the aforementioned ingenious structural design, the prepared substrate exhibits superior SERS performance: its dense yet rough multilayered silver nanostructure generates high-density electromagnetic field-enhancing "hot spots"; experiments have confirmed its extremely strong signal enhancement effect on probe molecules such as 4-ATP, and due to its uniform and controllable structure, the signal reproducibility (between sites and batches) of the substrate is also significantly improved. More importantly, the substrate exhibits outstanding physicochemical stability and reusability. The mechanical bond between the silver nanoparticles and AAO formed by capillary forces is very strong; even after repeated vigorous washing with solvents of different polarities such as water and acetonitrile, the SERS signal intensity does not show significant attenuation. This demonstrates its key advantages of being washable and reusable, effectively solving the core problem of the lack of durability of substrates prepared by conventional solution methods, and significantly reducing the cost per detection.
[0016] 4. This invention features a simple and low-cost process, requiring no complex or expensive equipment. It can be completed at room temperature and pressure, facilitating large-scale preparation. Furthermore, this method can be extended to the composite of gold and other precious metal nanoparticles with porous substrates, demonstrating broad applicability. In summary, this invention, through a clever physicochemical process, combines the advantages of AAO template nanostructures with solution processing, providing a new approach for preparing high-performance, highly stable, and reusable SERS substrates. This has significant application value in trace detection and biosensing. Attached Figure Description
[0017] Figure 1 This is a cross-sectional SEM image of the SERS substrate surface after the Ag film was self-assembled on the surface of the substrate prepared in Example 1.
[0018] Figure 2 The image shows the electromagnetic field simulation results of the SERS substrate prepared in Example 1.
[0019] Figure 3 The image shows the SERS substrate washing test signal obtained in Example 1. The first image represents the initial signal, and the signal did not significantly decrease after five water washes and five acetonitrile washes.
[0020] Figure 4 This is a SEM image of Ag nanoparticles on the surface of the SERS substrate obtained in Example 1.
[0021] Figure 5 The above is a comparison of the detection signals using different substrates in Example 1 and Example 2, where red represents the detection signal of the substrate in Example 1 and blue represents the signal detected by the substrate in Example 2.
[0022] Figure 6 The image shown is a SEM image of the AAO membrane surface without pretreatment in Example 3, which shows that some areas failed to bind with Ag. Detailed Implementation
[0023] The present invention will now be described in detail with reference to specific embodiments.
[0024] This invention provides a method for preparing a high-performance, reusable SERS substrate. The core of this method is as follows: First, a purification and concentration technique based on liquid-liquid interface separation is used to treat the silver nanoparticle sol. Then, the purified high-concentration silver nanoparticle solution is assembled on the porous surface of an AAO membrane using the capillary force-guided self-assembly principle to form a dense multilayer silver nanostructure membrane.
[0025] The method specifically includes the following steps: Preparation and purification of silver nanoparticle sol: Silver nanoparticle sol was prepared using a conventional chemical reduction method (such as sodium citrate reducing silver nitrate). The obtained sol was transferred to centrifuge tubes and washed by centrifugation. 15-20 ml of the final silver nanoparticle dispersion (at which point some silver nanoparticles deposited or aggregated on the tube wall due to centrifugation) was taken each time and allowed to stand together with a mixed solution of n-hexane and ethanol. Since ethanol and n-hexane are partially miscible, a dynamic liquid-liquid interface system is formed. Based on their surface properties (such as surface ligands and charge), silver nanoparticles tend to accumulate at the interface between two phases or in one phase, thereby achieving separation from excess reactants and byproducts, and obtaining a high-concentration, well-dispersed silver nanoparticle suspension (usually enriched at the interface). After carefully extracting the silver nanoparticle suspension enriched at the interface layer, the bottom layer of mixed solution is pipetted out and used to rinse the centrifuge tube wall to recover the attached particles. Finally, this rinsing solution is poured back into the previous hexane-ethanol-water mixture and allowed to stand for separation again, which can significantly improve the recovery rate of silver nanoparticles.
[0026] Preparation of AAO@Ag nanostructure substrate: A pretreated AAO membrane (with enhanced surface hydrophilicity) is placed horizontally. The interfacial layer or corresponding liquid phase enriched with silver nanoparticles after liquid-liquid separation is extracted and added dropwise or impregnated onto the AAO membrane surface. During the natural evaporation and drying process, due to the abundant nanoscale pores of the AAO membrane, the capillary force generated by the solvent evaporation within the pores guides the silver nanoparticles to migrate, aggregate, and tightly stack towards the inner walls of the pores and the membrane surface. By controlling the number of drops or the concentration, a uniform and dense multilayered silver nanoparticle coating layer, i.e., a silver film, can be formed on the AAO surface and within the near-surface pores.
[0027] To further understand the present invention, the method provided by the present invention will be described in detail below with reference to the embodiments. The scope of protection of the present invention is not limited by the following embodiments.
[0028] Example 1 (1) Preparation of silver nanoparticle sol: Heat 100 mL of 5 mM silver nitrate solution to boiling, quickly add 3 wt% sodium citrate solution, continue to react and heat until the solution turns golden yellow, then magnetically suspend and stir for half an hour to obtain gray-green silver sol.
[0029] (2) Liquid-liquid separation and purification: The obtained solution was transferred to a centrifuge tube and centrifuged at 10,000 rpm for 20 minutes. The supernatant was discarded, and the solution was resuspended in deionized water. The centrifugation and washing were repeated three times. The washed and purified silver nanoparticles were dispersed in deionized water to obtain a silver nanoparticle dispersion (15-20 mL each time). The silver nanoparticle dispersion was mixed with an equal volume of n-hexane-ethanol solution and allowed to stand for separation to form a three-phase system. The silver nanoparticles were selectively enriched at the liquid-liquid interface layer. After removing the interfacial suspension, the lower mixed solution was used to rinse the centrifuge tube wall to recover residual particles. The rinsing solution was returned to the reducing system for a second separation by standing, and the interfacial suspensions were combined. The average diameter of the silver nanoparticles was determined to be approximately 100 nm. Figure 4 As shown.
[0030] (3) AAO substrate pretreatment: The AAO membrane (pore size about 30 nm, pore spacing about 65 nm, thickness about 50 μm, diameter 13 mm) was first ultrasonically cleaned with ethanol and deionized water, dried with nitrogen, and then treated with oxygen plasma for 30 min to achieve deep cleaning and superhydrophilization; finally, the activated membrane was immersed in an aminosilane solution for 1-2 hours for chemical modification (the aminosilane solution is an organic solvent solution of 3-aminopropyltrimethoxysilane (APTMS)). After cleaning and heat treatment by baking at 120°C in a vacuum drying oven for 1-2 hours, an AAO substrate with amino (-NH2) functional groups covalently bonded to its surface can be obtained. This modified substrate can provide an ideal active interface for the subsequent viable loading of silver nanoparticles and the immobilization of biological probes.
[0031] (4) Self-assembly into a membrane: The purified silver nanoparticle suspension was uniformly dropped onto the surface of the pretreated AAO membrane and dried at room temperature in a clean environment. This process was repeated 4 times, with 150 mL added each time to obtain a silver nanoparticle layer of the desired thickness.
[0032] The morphology of the prepared AAO@Ag substrate was characterized using scanning electron microscopy (SEM), and the results are shown in the figure. Figure 1 , Figure 1 The images show that silver nanoparticles densely cover the surface and pores of the AAO membrane, forming a rough multilayer structure, confirming the coverage state of silver nanoparticles on the AAO surface and the formation of the multilayer structure.
[0033] Electromagnetic field simulation was performed using the finite-difference time-domain (FDTD) method. The results are shown in [Figure / Reference]. Figure 2 , Figure 2 This indicates that the structure can generate a strong local electromagnetic field enhancement under excitation light irradiation.
[0034] After modifying the surface of Ag nanoparticles with the probe molecule 4-aminothiophenol (4-ATP), SERS detection was performed to verify its signal enhancement effect. The results are shown in the figure. Figure 3 The results showed that a strong SERS signal was obtained under 532 nm laser excitation. The substrate with the measured SERS signal was then sequentially immersed in deionized water and acetonitrile, and ultrasonically cleaned for a certain period of time. This constituted one cycle, and a total of five cycles were performed. After each cleaning and drying, the SERS signal was re-detected. The results showed that after five washes, the characteristic peak intensity of 4-ATP remained above 90% of the initial intensity, indicating that the silver nanoparticles were firmly bound to the AAO substrate, the substrate had good stability, and it could be repeatedly used for detection.
[0035] Example 2 In this embodiment, chemical deposition is used instead of the self-assembled film in Example 1. The AAO surface is first soaked in a divalent tin solution for 1 minute to allow it to "receive" the silver. Then, it is immersed in a weakly alkaline solution containing silver nitrate, ammonia, glucose, and formaldehyde at room temperature for half an hour to obtain the silver layer. Other operations are largely the same as in Example 1 and will not be repeated here. The silver layer obtained by chemical deposition lacks the gaps between the silver spheres in the silver sphere layer, resulting in a weaker local electromagnetic field and a correspondingly weaker SERS signal. Finally, signal detection of the substrate revealed a significantly lower signal than in Example 1, confirming this expectation. Specifically, as shown below... Figure 5 As shown.
[0036] Example 3 In this embodiment, the difference from Example 1 is that the AAO substrate is not pretreated. The purified silver nanoparticle suspension from Example 1 is uniformly dropped onto the surface of the untreated AAO membrane and dried at room temperature in a clean environment. This process is repeated 4 times, with 150 mL added each time to obtain a silver nanoparticle layer of the same thickness as in Example 1.
[0037] The morphology of the AAO@Ag substrate prepared in Example 3 was characterized using scanning electron microscopy (SEM). The results showed that although Ag could be adsorbed on the AAO surface, gaps appeared, and a complete and dense silver film could not be formed. Specifically, as shown in the figure... Figure 6 As shown.
[0038] The above description of the embodiments is only for illustrating the technical concept and features of the present invention. Its purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. Those skilled in the art can obviously easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative effort. Therefore, the above embodiments should not be used to limit the scope of protection of the present invention. All improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be covered within the scope of protection of the present invention.
Claims
1. A method for preparing a reusable SERS substrate, characterized in that, Includes the following steps: (1) Preparation of silver nanoparticle sol: Heat a silver nitrate solution of a certain concentration to boiling, quickly add sodium citrate solution, continue the reaction for a certain time, and when the solution turns golden yellow, suspend and magnetically stir until the solution gradually turns gray-green, stop heating, and obtain silver sol; (2) Liquid-liquid separation and purification: The obtained silver sol was centrifuged and washed with deionized water in a centrifuge tube, and then resuspended with deionized water to obtain a silver nanoparticle dispersion. The silver nanoparticle dispersion was poured into an equal volume of n-hexane-ethanol mixed solution, and the solution was allowed to stand for layering. The silver nanoparticles were selectively enriched in the liquid-liquid interface layer. (3) AAO substrate pretreatment: First, the AAO membrane is ultrasonically cleaned with ethanol and deionized water, dried with nitrogen, and then treated with an oxygen plasma cleaner to achieve deep cleaning and superhydrophilization; finally, the activated membrane is immersed in an aminosilane solution for chemical modification, and after cleaning and heat treatment, an AAO substrate with amino functional groups covalently bonded to the surface can be obtained. (4) Self-assembly into a film: Take the interfacial suspension from step (2) and drop it evenly onto the surface of the pretreated AAO membrane, and dry it at room temperature to obtain the SERS substrate.
2. The method for preparing a reusable SERS substrate according to claim 1, characterized in that: The concentration of the silver nanoparticle dispersion is approximately 1 mM, and the particle size of the silver nanoparticles is 80~120 nm.
3. The method for preparing a reusable SERS substrate according to claim 1, characterized in that: The volume ratio of hexane to ethanol in the hexane-ethanol mixed solution is 1:0.8~1.
2.
4. The method for preparing a reusable SERS substrate according to claim 1, characterized in that: The aminosilane solution is an organic solvent solution of 3-aminopropyltrimethoxysilane, and the AAO membrane is immersed in the solution for 1-2 hours.
5. The method for preparing a reusable SERS substrate according to claim 1, characterized in that: The heat treatment process involves baking at 120°C in a vacuum drying oven for 1-2 hours to cure the material.
6. The method for preparing a reusable SERS substrate according to claim 1, characterized in that: In step (4), the interfacial suspension can be added dropwise and dried multiple times to obtain a silver nanoparticle layer of the required thickness.