SERS (Surface Enhanced Raman Scattering) spectrum detection method based on molecular substitution method, SERS substrate and preparation method thereof

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) spectrum detection method based on a molecule replacement method, an SERS substrate and a preparation method of the SERS substrate, and belongs to the technical field of molecule detection. The method comprises the following steps that a nano-cavity structure is constructed on a substrate, the nano-cavity structure comprises at least two metal surfaces, at least one metal surface is provided with a molecular layer, and a nanoscale gap is formed between the metal surfaces. And placing the matrix in a target molecule solution for molecule replacement, and carrying out spectrum detection after molecule replacement is completed. According to the method, a molecular layer on the surface of a metal nanoparticle serves as a dynamically adjustable nano channel, and controllable migration and anchoring of target molecules into a plasmon nano cavity are achieved. The detection molecules entering the cavity can be used as nano channels to be replaced by new detection molecules, so that the structure is endowed with the capability of multi-round dynamic replacement, and the SERS substrate can be repeatedly used.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular detection, and in particular relates to a SERS spectrum detection method based on a molecular replacement method, a SERS substrate and a preparation method thereof. Background Art

[0002] Surface-enhanced Raman scattering (SERS) can detect molecules adsorbed on noble metal surfaces in monolayers and submonolayers, providing structural fingerprint information. It boasts advantages such as high sensitivity, high accuracy, fingerprint identification, and non-destructive testing, and is widely used in materials science, chemistry, nanoscience, nanotechnology, interdisciplinary chemistry, applied physics, spectroscopy, and biomedicine. The sensitivity and signal reproducibility of SERS spectroscopy are closely related to the type of substrate. Research has shown that metal substrates are key to SERS technology, with metals such as gold (Au), silver (Ag), and copper (Cu) serving as excellent SERS substrates. By manipulating the nanostructure and size of metal substrates, SERS substrates with varying selectivity and functionality can be prepared, enabling the detection and application of different target molecules.

[0003] Common SERS substrate configurations include the following categories: 1. Colloidal nanoparticles; 2. Nanostructured metal films; 3. Nanohole / nanogap arrays; 4. Template-assisted 3D nanostructures; 5. Nanoparticle-on-Mirror (NPoM).

[0004] In existing technologies, nanoparticle-mirror structures have been widely used to construct nano-optical cavities, which are particularly suitable for scenarios such as surface-enhanced Raman scattering (SERS), nonlinear optics, and molecular detection. This type of nanocavity structure is usually prepared by a bottom-up assembly method, that is, a monolayer is first formed on the surface of a metal mirror through self-assembly technology, or by transferring a two-dimensional material (such as graphene, hBN) as a spacer layer, and then metal nanoparticles are deposited on it to complete the cavity construction (see Figure 1 ).

[0005] However, existing technologies have the following key limitations in practical applications: (1) The structure is highly closed, which limits the way to introduce analytes. Since the cavity is completely closed during the assembly process, the substance to be detected must be pre-fixed on the metal film surface before the nanocavity is formed. This process is limited by the sample pretreatment method, molecular adsorption efficiency and the volatility of the deposition process, which increases the uncertainty of cavity construction and is not conducive to the consistency control and standardized application of the cavity structure. (2) It is impossible to achieve real-time or dynamic analyte monitoring. Since the analyte needs to be introduced before the cavity is constructed, the existing method does not support the dynamic introduction of external samples after the cavity is constructed, which makes it difficult to meet the real-time monitoring requirements of target molecules in flowing samples, reaction systems or environments. (3) The structure is not reusable and the material cost is high. The NPoM structure mostly uses precious metal materials such as gold and silver, and once the cavity is constructed, it is difficult to disassemble or clean and reuse it, resulting in material waste, significantly increasing the single-use cost of the sensor, and is not conducive to large-scale, low-cost commercial promotion. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a SERS spectral detection method, a SERS substrate and a preparation method thereof based on the molecular replacement method. The present invention adopts the "molecular replacement method" to construct a reusable SERS substrate. This method uses the surfactant or molecular layer on the surface of the metal nanoparticles as a dynamically adjustable "nanochannel" to achieve the controllable migration and anchoring of the target molecules into the plasmon nanocavity. The detection molecules entering the cavity can be replaced by new detection molecules as "nanochannels", giving the structure the ability of multiple rounds of dynamic replacement, thereby realizing the reusability of the SERS substrate. The open cavity construction method significantly improves the practicality of the structure, and can be combined with a microfluidic system to achieve high-sensitivity, real-time online detection of target molecules.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a SERS spectroscopy detection method based on a molecular replacement method, comprising the following steps: Constructing a nanocavity structure on a substrate, wherein the nanocavity structure comprises at least two metal surfaces, at least one of which is provided with a surfactant, and a nanoscale gap is formed between the metal surfaces; The substrate is placed in a target molecule solution for molecular replacement, and after the molecular replacement is completed, SERS spectrum detection is performed using a SERS spectrum detection device.

[0008] This method utilizes the enormous electromagnetic field enhancement generated by the nanocavity gap. Therefore, any nanocavity structure capable of forming an effective SERS "hotspot" can serve as the substrate for this method. A nanoscale gap (typically <10 nm) is formed between the gaps, which serves as the SERS "hotspot." Through a molecular replacement step, the initially modified surfactant molecules (whose SERS signal could interfere with detection) are effectively removed, allowing the target molecule to directly occupy the "hotspot" position with the strongest signal, significantly reducing background interference and improving the detection signal-to-noise ratio and specificity. This method is applicable to a variety of target molecules that can undergo displacement adsorption on metal surfaces (such as biomolecules, drug molecules, environmental pollutants, and explosive molecules). The design and selection of the nanocavity structure can be optimized based on the size and properties of the target molecule, as well as the desired enhancement factor. The key is to form a nanogap that can accommodate the target molecule and generate a strong localized electromagnetic field.

[0009] Preferably, the molecular layer comprises polyvinyl pyrrolidone, sodium citrate or cetyltrimethylammonium bromide. After the surfactant covering the surface of the nanoparticles is deposited, the surfactant molecules inside the cavity serve as "channels" for initial replacement.

[0010] Preferably, the target molecules in the target molecule solution include amino, thiol, or selenol groups, which can effectively and stably adsorb to metal surfaces. Such molecules generally have high metal binding energies and can effectively and stably adsorb to metal surfaces, providing favorable conditions for subsequent molecular replacement.

[0011] Preferably, after the first SERS spectrum detection is completed, the substrate can be placed in the next target molecule solution for molecule replacement and the SERS spectrum detection can be continued; by continuously replacing the target molecule solution, the substrate can be reused to complete continuous detection or dynamic monitoring.

[0012] The present invention achieves effective anchoring and detection of target molecules by gradually replacing existing surfactant molecules within the cavity, allowing them to enter the interstitial region of the nanocavity. Furthermore, by varying the sample's immersion time in the solution of detection molecules, the migration depth and spatial distribution of the detection molecules within the cavity interstitial region can be controlled, further enhancing the functional flexibility and detection performance of the cavity structure.

[0013] During the dynamic replacement process, the detection molecules that have been replaced into the gap of the nanocavity can also act as new "molecular channels" and be further dynamically replaced by subsequent molecules to be detected. The formed nanocavity structure is placed in a new solution of molecules to be detected. Driven by multiple factors such as the difference in binding energy between the molecules and the metal surface, the molecular concentration gradient, and the molecular diffusion effect, the subsequent molecules to be detected will gradually replace the original molecules embedded in the cavity and enter the interior of the nanocavity. Through this process, the progressive penetration and occupation of molecules can be achieved, thereby giving the nanocavity structure good multi-round reusability and supporting dynamic detection applications of different molecules to be detected.

[0014] As an advantage, the method comprises the following steps: S1. The metal particles are mixed with a surfactant and a solvent to prepare a metal particle solution; S2. Drop the metal particle solution onto the surface of the ultra-smooth metal film, let it stand to allow the metal particles to deposit on the surface of the metal film, and then blow nitrogen to dry the sample to form an NPoM structure to obtain a SERS substrate.

[0015] The NPoM structure is a "nanoparticle-mirror" structure. It is essentially a highly controllable "hotspot" structure with a subnanometer-scale gap. The extremely narrow gap between the nanoparticle base and the metal mirror is often filled with a dielectric spacer or the target analyte molecules themselves.

[0016] The NPoM structure, constructed using molecular replacement, possesses open channels, allowing target molecules to enter the cavity in real time during the detection process, enabling dynamic monitoring of the target molecules in flowing samples, reaction systems, and environmental media. This feature overcomes the limitation of traditional methods, which lacks real-time monitoring, and provides a new technical path for in situ characterization, time-resolved analysis, and high-throughput sensing.

[0017] The reusability of the cavity structure is also achieved, reducing costs. The molecular replacement method utilizes a controllable molecular replacement process, allowing for multiple rounds of dynamic replacement of the detection molecules within the cavity, endowing the NPoM structure with excellent reusability. Users can switch between different test molecules through simple solution processing, avoiding the need for scrapping and rebuilding the cavity structure. This significantly reduces the frequency of precious metal material use and the overall sensor cost, promoting low-cost, large-scale commercial applications.

[0018] More preferably, the metal particles are made of gold, silver, copper, platinum or aluminum.

[0019] Preferably, the particle size of the metal particles is nanometer or micrometer scale; The particle size of nano-scale metal particles is 40~150nm; The particle size of micron-sized metal particles is 1~10μm.

[0020] More preferably, the metal particles are in the shape of spheres, rods, stars or cubes.

[0021] More preferably, the surfactant includes polyvinyl pyrrolidone, sodium citrate or cetyltrimethylammonium bromide.

[0022] Further preferably, the solvent includes deionized water, ethanol, PBS buffer, TBS buffer, which are common solvents commonly used as dispersion media in chemical or biological sensing scenarios.

[0023] Preferably, the concentration of the metal particle solution is 0.001-0.02 mg / mL.

[0024] Preferably, the amount of the metal particle solution added to the surface of the ultra-smooth metal film is 20-50ul / cm 2 .

[0025] Further preferably, the material of the ultra-smooth metal film is gold, silver, chromium, or titanium; The roughness of the ultra-smooth metal film is 0.2-0.5 nm.

[0026] More preferably, the standing time is 10 min.

[0027] In a third aspect, the present invention provides a reusable SERS substrate prepared by the above-mentioned preparation method.

[0028] Contains at least the following beneficial technical effects: While maintaining the consistency of the physical properties of the nanocavity structure, the molecular replacement method described in the present invention has significant application advantages compared to traditional bottom-up assembly methods, including: achieving local electric field enhancement of up to several hundred times at the subwavelength scale, effectively enhancing the interaction strength between light and matter; having an adjustable nanogap size, and a structurally stable and highly repeatable local electromagnetic mode.

[0029] The open cavity structure enhances the flexibility of analyte introduction. Molecular replacement, through a dynamic displacement process, enables target molecules to freely migrate into the nanocavity through the liquid phase after the cavity structure is completed. This significantly overcomes the limitations of traditional NPoM structures, which are "strongly closed and require pre-embedded analytes." This method eliminates the need for complex sample pretreatment and adsorption optimization, reduces dependence on process conditions, improves the consistency and standardization of the cavity construction process, and facilitates mass production and practical application.

[0030] This invention supports real-time, dynamic analyte monitoring. The NPoM structure, constructed using molecular replacement, possesses open channels, allowing target molecules to enter the cavity in real time during the detection process, enabling dynamic monitoring of the target molecule in flowing samples, reaction systems, and environmental media. This feature overcomes the limitation of traditional methods, which often lack real-time monitoring, and provides a new technical path for in situ characterization, time-resolved analysis, and high-throughput sensing.

[0031] This invention achieves reusability of the cavity structure and reduces costs. The molecular replacement method utilizes a controllable molecular replacement process, allowing for multiple rounds of dynamic replacement of the detection molecule within the cavity, endowing the NPoM structure with excellent reusability. Users can switch between different test molecules through simple solution processing, avoiding the need for scrapping and rebuilding the cavity structure. This significantly reduces the frequency of precious metal material use and the overall sensor cost, promoting low-cost, large-scale commercial applications.

[0032] This invention supports the construction of micron-scale cavities, broadening the scope of application. The molecular replacement method is not only applicable to the construction of traditional nanoscale NPoM cavities, but can also be extended to micron-scale plasmon cavity structures. By introducing a dynamic molecular replacement process between micron-scale metal nanostructures (such as gold hexagonal disks) and gold films, it is possible to achieve controlled migration and efficient anchoring of target molecules within large-scale cavities. Raman mapping technology can clearly characterize the spatial evolution of the molecular replacement process, further demonstrating that this method has excellent scale adaptability and molecular permeation control capabilities, providing a new technical path for large-area sensor design, multi-scale characterization, and in situ analysis of complex systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the SERS substrate structure of the traditional nanoparticle-mirror structure.

[0034] Figure 2 Schematic diagram of the SERS substrate with nanoparticle-mirror structure prepared in the present invention.

[0035] Figure 3 This is a diagram showing the effects of multiple rounds of molecular replacement in Experimental Example 1.

[0036] Figure 4 This is a diagram showing the effects of multiple rounds of molecular replacement in Experimental Example 2.

[0037] Figure 5 This is a diagram showing the effect of using micron-sized metal particles to prepare the SERS substrate in Experimental Example 3. DETAILED DESCRIPTION

[0038] Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative work shall fall within the scope of protection of the present invention. The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0039] The roughness of the ultra-smooth gold film used in the following examples is 0.4 nm.

[0040] All reagents and materials unless otherwise specified were commercially available.

[0041] Example 1 Gold nanoparticles with a diameter of 150 nm and polyvinyl pyrrolidone on their surface were prepared with ethanol to form a uniform dispersion with a mass concentration of 0.002 mg / mL. Then, 50 μl of the dispersion was dropped onto the surface of a square ultra-smooth gold film with a side length of 1 cm and a thickness of 10 nm. The sample was allowed to stand for 10 minutes and then dried with nitrogen to obtain a SERS substrate with an NPoM structure.

[0042] Example 2 Gold nanoparticles with a diameter of 40 nm and polyvinyl pyrrolidone on their surface were prepared with ethanol to form a uniform dispersion with a mass concentration of 0.01 mg / mL. Then, 20 μl of the dispersion was dropped onto the surface of a square ultra-smooth gold film with a side length of 1 cm and a thickness of 10 nm. The sample was allowed to stand for 10 minutes and then dried with nitrogen to obtain a SERS substrate with an NPoM structure.

[0043] Example 3 Gold nanoparticles with a diameter of 5 μm and a surface-coated polyvinyl pyrrolidone were prepared with ethanol to form a uniform dispersion with a mass concentration of 0.02 mg / mL. Then, 30 μl of the dispersion was dropped onto the surface of a square ultra-smooth gold film with a side length of 1 cm and a thickness of 10 nm. The sample was allowed to stand for 10 minutes and then dried with nitrogen to obtain a SERS substrate with an NPoM structure.

[0044] Example 4 Silver hexagonal disk microplates with a diameter of 9 μm and a surface coated with polyvinyl pyrrolidone (PVP) were prepared into a uniform dispersion with a mass concentration of 0.002 mg / mL. The dispersion was then dropped onto the surface of a square ultra-smooth gold film with a side length of 1 cm and a thickness of 10 nm. The sample was allowed to stand for 10 minutes and then dried with nitrogen to obtain a SERS substrate with an NPoM structure.

[0045] Experimental Example 1 1) The SERS substrate prepared in Example 1 was placed in a 1 mmol / L solution of methylene blue (MB) molecules and allowed to stand for 2 hours. The amino groups in the MB molecules form a strong chemical bond with the gold surface, with a binding energy higher than that of polyvinyl pyrrolidone. Consequently, during this dynamic replacement process, the MB molecules spontaneously displaced the existing surfactant molecules within the cavity, forming a monolayer structure within the nanocavity gap. After treatment, the sample was removed, rinsed with ethanol, dried with nitrogen, and set aside. At this point, the "nanochannel" within the cavity was composed of MB molecules.

[0046] The process of preparing the molecular channel of NPoM structure is as follows: Figure 2 As shown, the NPoM structure prepared in Example 1 was immersed in a molecular solution to further prepare a "nanochannel" composed of molecules inside the cavity.

[0047] 2) The prepared sample is placed under the sample stage of a microscope. A 785-nanometer laser is selected and focused on the location of the nanoparticles through an objective lens. The scattered signal is collected through the same objective lens and detected using a confocal Raman microscope (HORIBA LabRAM Odyssey) to obtain the Raman spectrum.

[0048] 3) The NPoM sample, with methylene blue as the "channel" molecule, was re-introduced into a 1 mmol / L solution of phenylselenol (BSe) molecules and allowed to stand for 2 hours. Due to the difference in binding energies between the phenylselenol molecules, methylene blue, and the metal particle surface, as well as the combined effects of the solution concentration gradient and molecular diffusion, the phenylselenol molecules spontaneously replaced the existing methylene blue molecules in the cavity, forming a new monolayer. After this treatment, the sample was rinsed with ethanol and dried with nitrogen, and then set aside. At this point, the "nanochannels" within the cavity were composed of phenylselenol molecules.

[0049] Then, by placing the sample in a 1 mmol / L biphenyl-4-thiol (BPT) molecular solution, the dynamic replacement of phenylselenol inside the cavity by BPT molecules was achieved.

[0050] The above molecular replacement process can be dynamically controlled in multiple rounds in the same way, and each replacement process is characterized by Raman spectroscopy to verify the molecular replacement effect and the change in molecular distribution in the cavity. The test results are as follows: Figure 3 As shown. The Raman characteristic peaks of each molecule are detected by spectroscopy and Figure 3The area marked by the columnar shaded area in the figure. Experimental results demonstrate that these probe molecules can effectively replace the original channel molecules and successfully enter the nanocavity, producing clear Raman response signals. This fully demonstrates the stability and controllability of the nanocavity system prepared by the replacement method in terms of molecular replacement and reuse, and further demonstrates the potential of this type of plasmonic nanocavity structure in multi-cycle molecular detection and dynamic control applications.

[0051] Experimental Example 2 1) The SERS substrate prepared in Example 1 was placed in a 0.1 mmol / L biphenyl-4-thiol (BPT) molecular solution. The sulfur group in the biphenyl-4-thiol molecule can form a strong chemical bond with the gold surface. Therefore, during this dynamic replacement process, the biphenyl-4-thiol molecules can gradually replace the original surfactant molecules in the cavity, and finally form a monolayer structure in the nanocavity gap.

[0052] 2) Place the substrates in the solution for 5 seconds, then take out the samples, wash them with ethanol, blow dry them with nitrogen, and set aside.

[0053] 3) Place the prepared sample under the sample stage of the microscope, select a 785 nm wavelength laser, align it with the objective lens and focus it on the location of the nanoparticles, collect the scattered signal through the same objective lens, and obtain the Raman spectrum by a confocal micro-Raman spectrometer (LabRAM Odyssey of HORIBA) (see Figure 4 ).

[0054] 4) The matrix is ​​placed in the solution for different periods of time. The longer the time, the more BPT molecules are replaced, until the molecules reach saturated adsorption at the interface.

[0055] Repeat steps 2) and 3).

[0056] The above molecular replacement process can be dynamically regulated for multiple rounds according to different replacement times, and each replacement process is characterized by Raman spectroscopy to verify the molecular replacement effect. The test results are as follows: Figure 4As shown in the figure, the signal is weak within a short period (5–30 s), with only a few characteristic peaks observed. A clear spectrum gradually forms in the intermediate stage (300–900 s). Finally, after a long treatment period (7200 s), the spectrum intensity approaches saturation, with clear and well-resolved peaks, indicating that the BPT molecules within the cavity have reached a stable adsorption state. These results demonstrate that BPT molecules can gradually replace the existing surfactant molecules in the NPOM structure cavity by forming strong sulfur-gold bonds with the gold surface. During continuous immersion, the Raman signal enhancement trend is highly consistent with the molecular replacement process, verifying that the system supports a time-controlled molecular replacement mechanism and can be dynamically monitored in real time using SERS. Longer replacement time increases the surface coverage of BPT molecules, ultimately forming a stable, dense monolayer within the cavity, providing an experimental basis for the realization of a highly sensitive and controllable molecular detection platform.

[0057] Experimental Example 3 The SERS substrate prepared in Example 4 was placed in a 1 mmol / L BTP molecule solution, and the dynamic molecular replacement process was achieved by adjusting the immersion time. By using the surface enhanced Raman spectroscopy (SERS) characteristic peak mapping technology, the spatial boundaries during the molecular replacement process can be clearly identified and the evolution of the replacement process can be monitored until the original ligand molecules in the cavity are completely replaced by the target molecules. Figure 5 Optical imaging of the sample Figure 5 As shown in a. The initial stage of molecular replacement ( Figure 5 b), the Raman signal enhancement is obvious at the edge of the cavity, indicating that BPT molecules are preferentially replaced from the outer area of ​​the microcavity; as the immersion time increases ( Figure 5 c in Figure 5 In the d in the figure, the signal gradually expands toward the center of the cavity, indicating that the molecules gradually penetrate into the cavity from the periphery; the final stage ( Figure 5 (f) The signal distribution in the entire microcavity area tends to be uniform, and the replacement tends to be saturated; Figure 5 Figure g shows a typical Raman spectrum of the region where the replacement is complete, with multiple characteristic peaks of BPT clearly discernible. These results demonstrate that BPT molecules, through interaction with the metal surface, gradually replace the original ligand molecules in the cavity between the silver microsheet and the gold film, forming a stable monolayer structure. Raman mapping successfully reveals the spatial boundary migration characteristics of the molecular replacement process and provides a means to visualize and track the dynamic molecular penetration behavior.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A SERS spectroscopy detection method based on molecular replacement method, characterized in that: The following steps are involved: Constructing a nanocavity structure on a substrate, wherein the nanocavity structure comprises at least two metal surfaces, at least one of which has a molecular layer, and a nanoscale gap is formed between the metal surfaces; The substrate is placed in a target molecule solution for molecular replacement, and after the molecular replacement is completed, SERS spectrum detection is performed using a SERS spectrum detection device.

2. The SERS spectrum detection method according to claim 1, characterized in that: The nanocavity structure is a particle-mirror structure.

3. The SERS spectrum detection method according to claim 1, wherein: The molecular layer is polyvinyl pyrrolidone.

4. The SERS spectrum detection method according to claim 1, characterized in that: The target molecules in the target molecule solution include amino groups or selenol groups, which can effectively form stable adsorption molecules on the metal surface.

5. The SERS spectrum detection method according to claim 1, characterized in that: After the first SERS spectrum detection is completed, the matrix can be placed in the next target molecule solution for molecule replacement and the SERS spectrum detection can be continued; by continuously replacing the target molecule solution, the matrix can be reused to complete continuous detection or dynamic monitoring.

6. A method for preparing a reusable SERS substrate, characterized in that: The following steps are involved: S1. The metal particles are mixed with a surfactant and a solvent to prepare a metal particle solution; S2. Drop the metal particle solution onto the surface of the ultra-smooth metal film, let it stand to allow the metal particles to deposit on the surface of the metal film, and then blow nitrogen to dry the sample to form an NPoM structure to obtain a SERS substrate.

7. The preparation method according to claim 6, characterized in that The particle size of the metal particles is nanometer or micrometer; The particle size of nano-scale metal particles is 40~150nm; The particle size of micron-sized metal particles is 1~10μm.

8. The preparation method according to claim 6, characterized in that The concentration of the metal particle solution is 0.002-0.02 mg / mL.

9. The preparation method according to claim 6, characterized in that The material of the ultra-smooth metal film is gold; The roughness of the ultra-smooth metal film is 0.4 nm.

10. A reusable SERS substrate, characterized in that: The invention is prepared by the preparation method according to any one of claims 6 to 9.

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