SERS spectrum detection method based on molecular replacement method, sers substrate and preparation method thereof

Abstract

The application discloses a SERS spectrum detection method based on a molecular replacement method, a SERS substrate and a preparation method thereof, and belongs to the technical field of molecular detection. The application comprises the following steps: constructing a nano-cavity structure on a substrate, wherein the nano-cavity structure comprises at least two metal surfaces, at least one of the metal surfaces is provided with a molecular layer, and a nanoscale gap is formed between the metal surfaces. The substrate is placed in a target molecule solution to perform molecular replacement, and spectrum detection is performed after the molecular replacement is completed. The method takes the molecular layer on the surface of metal nanoparticles as a dynamically adjustable 'nanochannel', realizes controllable migration and anchoring of target molecules into a plasmonic nano-cavity, and the detection molecules entering the cavity can be replaced by new detection molecules as 'nanochannels', so that the structure has the ability of multiple rounds of dynamic replacement, and the SERS substrate can be reused.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular detection, and particularly relates to a SERS spectrum detection method based on a molecular replacement method, a SERS substrate and a preparation method thereof. BACKGROUND

[0002] Surface-enhanced Raman scattering (SERS) can detect a monomolecular layer and a submonomolecular layer of molecules adsorbed on a noble metal surface and provide fingerprint structure information of the molecules, and has advantages of high sensitivity, high accuracy, fingerprint identification and nondestructive detection, and is widely applied in fields of material science, chemistry, nanoscience, nanotechnology, multidisciplinary cross-chemistry, applied physics, spectroscopy and biomedical science. The sensitivity and signal reproducibility of SERS spectrum are closely related to the type of substrate. Studies have shown that a metal substrate is the key to SERS technology, and metals such as gold (Au), silver (Ag) and copper (Cu) are good SERS substrates, and by controlling the nanostructure and size of the metal substrate, SERS substrates with different selectivity and functionality can be prepared, so as to realize 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 the prior art, nanoparticle-mirror structures have been widely used to construct nanooptical cavities, and are particularly suitable for scenarios such as surface-enhanced Raman scattering (SERS), nonlinear optics and molecular detection. Such nanocavity structures are usually prepared by a bottom-up assembly method, that is, a monomolecular layer is first formed on the surface of a metal mirror by self-assembly technology, or a two-dimensional material (such as graphene, hBN) is transferred as a spacer layer, and then metal nanoparticles are deposited on the top to complete the cavity construction (see Figure 1 ).

[0005] However, the prior art has the following key limitations in practical applications:

[0006] (1) The structure is strong in sealing, and the introduction of analyte is limited. Since the cavity is completely sealed during assembly, the detected substance must be fixed on the metal film surface before the nanocavity is formed. This process is limited by sample pretreatment method, molecular adsorption efficiency and volatility of 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) Real-time or dynamic analyte monitoring cannot be achieved. Since the analyte needs to be introduced before the cavity is constructed, the existing method does not support dynamic introduction of external samples after the cavity is constructed, which makes it difficult to meet the real-time monitoring needs of target molecules in flowing samples, reaction systems or environment. (3) The structure cannot be reused, and the material cost is high. The NPoM structure mainly uses noble metal materials such as gold and silver, and the cavity is difficult to disassemble or clean for reuse after being constructed, which causes material waste and significantly increases the single-use cost of the sensor, which is not conducive to large-scale, low-cost commercial promotion. SUMMARY

[0007] Therefore, the present application aims to provide a SERS spectrum detection method based on a molecular replacement method, a SERS substrate and a preparation method thereof. The present application uses a "molecular replacement method" to construct a reusable SERS substrate. The method uses the surfactant or molecular layer on the surface of the metal nanoparticles as a dynamic adjustable "nanochannel" to realize the controllable migration and anchoring of target molecules into the plasmonic nanocavity. The detection molecules entering the cavity can be replaced by new detection molecules as "nanochannels", which endows the structure with the ability of multiple dynamic replacement, realizing the reusability of the SERS substrate. The open cavity construction method significantly improves the practicability of the structure, which can realize high-sensitivity and real-time online detection of target molecules combined with a microfluidic system.

[0008] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0009] In the first aspect, the present application provides a SERS spectrum detection method based on a molecular replacement method, comprising the following steps:

[0010] Constructing a nanocavity structure on the substrate, wherein the nanocavity structure comprises at least two metal surfaces and at least one metal surface is provided with a surfactant, and a nanoscale gap is formed between the metal surfaces;

[0011] Placing the substrate in a target molecule solution for molecular replacement, and then performing SERS spectrum detection by a SERS spectrum detection device after the molecular replacement is completed.

[0012] The present application utilizes the great electromagnetic field enhancement generated by the nanocavity gap. Therefore, any nanocavity structure capable of forming an effective SERS "hot spot" can be used as the substrate of the present method. A nanoscale gap (usually <10 nm) is formed in between, and the gap region is the SERS "hot spot". Through the molecular replacement step, the initially modified surfactant molecules (whose SERS signal may interfere with detection) are effectively removed, allowing the target molecules to directly occupy the "hot spot" position with the strongest signal, thereby significantly reducing background interference and improving detection signal-to-noise ratio and specificity. It is suitable for various target molecules (such as biomolecules, drug molecules, environmental pollutants, explosive molecules, etc.) that can be displaced and adsorbed on metal surfaces. The design and selection of nanocavity structures can be optimized according to the size, properties of the target molecules, and the required enhancement factor. The core is to form a nanogap that can accommodate the test molecules and produce a strong local electromagnetic field.

[0013] As a preferred, the molecular layer includes polyvinylpyrrolidone, sodium citrate or cetyltrimethylammonium bromide. The surfactant covered on the surface of the nanoparticle, after deposition, the surfactant molecules inside the cavity serve as the initial replacement "channel".

[0014] As a preferred, the target molecules of the target molecule solution include amino, mercapto or selenol groups, which can effectively form stable adsorption with the metal surface. Such molecules usually have high metal binding energy and can effectively form stable adsorption with the metal surface, providing good conditions for subsequent molecular replacement.

[0015] As a preferred, after the first SERS spectrum detection is completed, the substrate can be placed in the next target molecule solution for molecular replacement and continue SERS spectrum detection; by continuously replacing the target molecule solution substrate, the substrate can be repeatedly used to complete continuous detection or dynamic monitoring.

[0016] The present application gradually replaces the original surfactant molecules in the cavity with target molecules, and enters the nanocavity gap region, to achieve effective anchoring and detection of target molecules. In addition, by changing the soaking time of the sample in the detection molecule solution, the migration depth and spatial distribution of the probe molecules in the cavity gap can be controlled and adjusted, further enhancing the functional flexibility and detection performance of the cavity structure.

[0017] In the above dynamic displacement process, the detection molecules that have been displaced into the nanocavity gap can also act as new "molecular channels" and be further dynamically replaced by subsequent molecules to be detected. By placing the formed nanocavity structure in a new solution of molecules to be detected, under the driving of multiple factors such as the difference in binding energy between the molecules and the metal surface, the concentration gradient of the molecules, and the diffusion of the molecules, the subsequent molecules to be detected will gradually replace the original molecules embedded in the cavity and enter the nanocavity. Through this process, the progressive penetration and occupation of molecules can be achieved, thereby endowing the nanocavity structure with good multi-round reusability and supporting dynamic detection of different molecules to be detected.

[0018] As preferred, the method comprises the following steps:

[0019] S1. mixing metal particles with a surfactant and a solvent to prepare a metal particle solution;

[0020] S2. dropping the metal particle solution onto the surface of the ultra-smooth metal film, depositing the metal particles on the surface of the metal film by standing, and then drying the sample by nitrogen blowing to form an NPoM structure to obtain a SERS substrate.

[0021] The NPoM structure is a "nanoparticle-mirror" structure. It is essentially a highly controllable "hot spot" structure with a sub-nanometer gap. There is a very narrow gap between the bottom of the nanoparticle and the metal mirror, and the gap is often filled with a dielectric spacer layer or the target analyte molecules themselves.

[0022] The NPoM structure constructed by the molecular replacement method has an open channel, allowing target molecules to enter the cavity in real time during the detection process, and realizing the dynamic monitoring of molecules to be detected in flowing samples, reaction systems and environmental media. This feature breaks through the defect of "unable to realize real-time monitoring" of traditional methods, and provides a new technical path for in-situ characterization, time-resolved analysis and high-throughput sensing.

[0023] Meanwhile, the reusability of the cavity structure is realized, and the cost is reduced. The molecular replacement method uses a controllable molecular displacement process, so that the detection molecules in the cavity can be dynamically replaced for multiple rounds, and the NPoM structure has good reusability. Users can switch different molecules to be detected through simple solution treatment, avoiding the scrapping and reconstruction of the cavity structure, significantly reducing the use frequency of noble metal materials and the overall sensor cost, and being conducive to promoting low-cost, large-scale commercial applications.

[0024] Further preferably, the metal particle material is gold, silver, copper, platinum, or aluminum.

[0025] As preferred, the particle size of the metal particles is nanoscale or micrometer scale.

[0026] The particle size of the nanoscale metal particles is 40-150 nm.

[0027] The micrometer-sized metal particles have a particle size of 1-10 μm.

[0028] Further preferably, the metal particles have a shape of a sphere, a rod, a star or a cube.

[0029] Further preferably, the surfactant comprises polyvinylpyrrolidone, sodium citrate or cetyltrimethylammonium bromide.

[0030] Further preferably, the solvent comprises deionized water, ethanol, PBS buffer, TBS buffer, common solvents commonly used in chemical or biological sensing scenarios as dispersion medium.

[0031] As preferred, the concentration of the metal particle solution is 0.001-0.02 mg / mL.

[0032] As preferred, the amount of the metal particle solution dropped onto the surface of the ultra-smooth metal film is 20-50 ul / cm 2 .

[0033] Further preferably, the material of the ultra-smooth metal film is gold, silver, chromium, titanium.

[0034] The roughness of the ultra-smooth metal film is 0.2-0.5 nm.

[0035] Further preferably, the standing time is 10 min.

[0036] In a third aspect, the present application provides a reusable SERS substrate prepared by the preparation method described above.

[0037] At least the following beneficial technical effects are achieved:

[0038] The molecular replacement method of the present application has significant application advantages compared to the traditional bottom-up assembly method while maintaining the consistency of the physical properties of the nanocavity structure, including: achieving a local electric field enhancement of up to several hundred times at a subwavelength scale, effectively enhancing the interaction strength between light and matter; having adjustable nanogap size, and stable structure with good repeatability of local electromagnetic mode.

[0039] Through the open cavity structure, the flexibility of analyte introduction is improved. The molecular replacement method enables the target molecules to freely migrate to the nanocavity interior through the liquid phase environment after the cavity structure is completed through a dynamic displacement process, significantly breaking through the limitations of traditional NPoM structures with strong "closure" and the need for pre-embedded analytes. This method does not require complex sample pretreatment and adsorption optimization processes, reducing the dependence on process conditions, improving the consistency and standardization level of the cavity construction process, and facilitating batch production and practical application promotion.

[0040] The present application supports real-time, dynamic analyte monitoring. The NPoM structure constructed by the molecular replacement method has an open channel, allowing target molecules to enter the cavity in real time during the detection process, achieving dynamic monitoring capability of the target molecules in flowing samples, reaction systems and environmental media. This feature breaks through the defect of "unable to realize real-time monitoring" of traditional methods, providing a new technical path for in-situ characterization, time-resolved analysis and high-throughput sensing.

[0041] The present application realizes the reusability of the cavity structure, reducing the cost. The molecular replacement method uses a controllable molecular replacement process, allowing the detection molecules inside the cavity to be dynamically replaced in multiple rounds, giving the NPoM structure good reusability. Users can switch different target molecules through simple solution processing, avoiding the scrapping and reconstruction of the cavity structure, significantly reducing the usage frequency of precious metal materials and the overall sensor cost, and facilitating the promotion of low-cost, large-scale commercial applications.

[0042] The present application supports the construction of micron-scale cavities, widening the application scale range. The molecular replacement method is not only suitable for the construction of traditional nanoscale NPoM cavities, but also can be extended to micron-scale plasmonic cavity structures. By introducing a dynamic molecular replacement process between micron-scale metal nanostructures (such as gold hexagonal plates) and gold films, controllable migration and efficient anchoring of target molecules in large-size cavities can be achieved. The use of Raman mapping technology can clearly characterize the spatial evolution during the molecular replacement process, further demonstrating that the method has good scale adaptability and molecular penetration control ability, providing a new technical path for large-area sensor design, multi-scale characterization and in-situ analysis of complex systems. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Schematic diagram of a traditional nanoparticle-mirror structure SERS substrate.

[0044] Figure 2 Schematic diagram of a nanoparticle-mirror structure SERS substrate prepared by the present application.

[0045] Figure 3 Effect diagram of multiple rounds of molecular replacement in Experimental Example 1.

[0046] Figure 4 Effect diagram of multiple rounds of molecular replacement in Experimental Example 2.

[0047] Figure 5 Effect diagram of the preparation of a SERS substrate using micron-scale metal particles in Experimental Example 3. DETAILED DESCRIPTION

[0048] All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative labor fall within the scope of the present application. The present application is described below with reference to specific embodiments, and it is to be understood that these embodiments are merely illustrative of, and not restrictive on, the present application.

[0049] The roughness of the ultrasmooth gold film used in the following examples was 0.4 nm.

[0050] Unless otherwise specified, reagents or materials were obtained from commercial sources.

[0051] Example 1

[0052] Gold nanoparticles with a diameter of 150 nm and polyvinylpyrrolidone on the surface were prepared into a uniform dispersion with a mass concentration of 0.002 mg / mL in ethanol, and then 50 ul of the dispersion was dropped onto the surface of a square ultrasmooth gold film with a side length of 1 cm and a thickness of 10 nm, and left to stand for 10 min. Subsequently, the sample was blown dry with nitrogen to obtain a SERS substrate with a NPoM structure.

[0053] Example 2

[0054] Gold nanoparticles with a diameter of 40 nm and polyvinylpyrrolidone on the surface were prepared into a uniform dispersion with a mass concentration of 0.01 mg / mL in ethanol, and then 20 ul of the dispersion was dropped onto the surface of a square ultrasmooth gold film with a side length of 1 cm and a thickness of 10 nm, and left to stand for 10 min. Subsequently, the sample was blown dry with nitrogen to obtain a SERS substrate with a NPoM structure.

[0055] Example 3

[0056] Gold nanoparticles with a diameter of 5 μm and polyvinylpyrrolidone on the surface were prepared into a uniform dispersion with a mass concentration of 0.02 mg / mL in ethanol, and then 30 ul of the dispersion was dropped onto the surface of a square ultrasmooth gold film with a side length of 1 cm and a thickness of 10 nm, and left to stand for 10 min. Subsequently, the sample was blown dry with nitrogen to obtain a SERS substrate with a NPoM structure.

[0057] Example 4

[0058] Silver hexagonal disc microparticles with a diameter of 9 um and polyvinylpyrrolidone (PVP) on the surface were prepared into a uniform dispersion with a mass concentration of 0.002 mg / mL, and then the dispersion was dropped onto the surface of a square ultrasmooth gold film with a side length of 1 cm and a thickness of 10 nm, and left to stand for 10 min. Subsequently, the sample was blown dry with nitrogen to obtain a SERS substrate with a NPoM structure.

[0059] Experimental Example 1

[0060] 1) The SERS substrate prepared in Example 1 was placed in a methylene blue (MB) molecule solution with a concentration of 1 mmol / L and left to stand for 2 hours. The amino group in the methylene blue molecule can form a strong chemical bond with the gold surface, and the bonding energy is higher than that of polyvinylpyrrolidone and the gold surface. Therefore, in this dynamic replacement process, the methylene blue molecules can spontaneously replace the original surfactant molecules in the cavity, and form a monolayer structure in the intercavity gap. After the treatment, the sample was taken out, washed with ethanol in sequence, dried with nitrogen, and ready for use. At this time, the "nanochannel" inside the cavity is composed of methylene blue molecules.

[0061] The process of preparing the molecular channel of the NPoM structure is shown in Figure 2 The NPoM structure prepared in Example 1 was immersed in a molecular solution to further prepare a "nanochannel" inside the cavity composed of molecules.

[0062] 2) The completed sample was placed under the sample stage of the microscope, a 785 nm wavelength laser was selected, and the laser was aligned and focused on the location of the nanoparticles through the objective lens. The scattered signal was collected through the same objective lens, and the Raman spectrum was detected by a confocal Raman spectrometer (LabRAM Odyssey of HORIBA Company).

[0063] 3) The NPoM sample with methylene blue molecules as "channel" molecules was re-placed in a benzene selenol (BSe) molecule solution with a concentration of 1 mmol / L and left to stand for 2 hours. Due to the difference in bonding energy between the benzene selenol molecule and the methylene blue and the metal particle surface, as well as the combined effects of the solution concentration gradient and molecular diffusion, the original methylene blue molecules in the cavity can be spontaneously replaced to form a new monolayer. After the treatment, the same ethanol washing and nitrogen drying were used in sequence, and ready for use. At this time, the "nanochannel" inside the cavity is composed of benzene selenol molecules.

[0064] Then, the sample was placed in a 1 mmol / L biphenyl-4-thiol (BPT) molecule solution to realize the dynamic replacement of BPT molecules for the benzene selenol inside the cavity.

[0065] The above-mentioned molecular replacement process can be carried out in the same way for multiple rounds of dynamic regulation, and each replacement process is characterized by Raman spectrum to verify the molecular replacement effect and the change of the distribution of molecules in the cavity. The detection results are shown in Figure 3 The Raman characteristic peaks of each molecule were detected by spectrum and shown in Figure 3The experimental results show that these probe molecules can effectively replace the original channel molecules and successfully enter the interior of the nanocavity to obtain clear Raman response signals. The stability and controllability of the nanocavity system prepared by the replacement method in terms of molecular replacement and reuse are fully verified, and the potential of such plasmonic nanocavity structures in multiple rounds of molecular detection and dynamic regulation applications is further illustrated.

[0066] Experimental Example 2

[0067] 1) The SERS substrate prepared in Example 1 was placed in a 0.1 mmol / L concentration of 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, so in 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.

[0068] 2) The substrate was placed in the solution for 5s, then the sample was taken out, washed with ethanol, and dried with nitrogen.

[0069] 3) The completed sample was placed under the sample stage of the microscope, a 785 nm wavelength laser was selected, and the laser was aligned and focused on the location of the nanoparticles through the objective lens. The scattered signal was collected through the same objective lens, and the Raman spectrum was detected by a confocal micro-Raman spectrometer (HORIBA LabRAM Odyssey) (see Figure 4 ).

[0070] 4) The substrate was placed in the solution for different times, the longer the time, the more BPT molecules were replaced, until the interface reached saturation adsorption.

[0071] Repeat steps 2) and 3).

[0072] The above molecular replacement process can be dynamically regulated for multiple rounds according to the replacement time, and each replacement process is characterized by Raman spectroscopy to verify the molecular replacement effect. The detection results are as follows: Figure 4The Raman signal is weak in a short time (5-30 s) and only partial characteristic peaks are observed; a clear spectrum is gradually formed in the middle stage (300-900 s); and the spectrum intensity tends to be saturated, the peak position is clear and well resolved after long time treatment (7200 s), indicating that the intracavity BPT molecules tend to be stable adsorption state. The above results show that BPT molecules can replace the original surfactant molecules in the NPOM structure cavity by forming a strong sulfur-gold bond with the gold surface. In the continuous immersion process, the enhancement trend of the Raman signal is highly consistent with the molecular replacement process, verifying that the system supports a time-controllable molecular replacement mechanism and can be dynamically monitored in real time by SERS. The longer the replacement time, the higher the surface coverage of BPT molecules, and finally a stable and dense monolayer is formed in the cavity, providing an experimental basis for realizing a high-sensitivity and controllable molecular detection platform.

[0073] Experimental Example 3

[0074] The SERS substrate prepared in Example 4 was placed in a solution of 1 mmol / L BTP molecules, and a dynamic molecular replacement process was achieved by adjusting the immersion time. The spatial boundary in the molecular replacement process can be clearly identified by the surface-enhanced Raman spectroscopy (SERS) characteristic peak mapping technology, 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, as shown in Figure 5 The optical image of the sample is shown in Figure 5 a. In the initial stage of molecular replacement (b of Figure 5 ), the Raman signal enhancement at the edge of the cavity is obvious, indicating that BPT molecules preferentially replace the molecules from the outer region of the microcavity; as the immersion time increases (c of Figure 5 , d of Figure 5 ), the signal gradually spreads to the center of the cavity, indicating that the molecules gradually penetrate into the inside of the cavity from the periphery; in the final stage (f of Figure 5 ), the signal distribution of the entire microcavity region tends to be uniform, and the replacement tends to be saturated; Figure 5 g shows the typical Raman spectrum of the replaced region, and the multiple characteristic peaks of BPT are clearly distinguishable. The above results show that BPT molecules can replace the original ligand molecules in the cavity between the silver microparticle and the gold film by interacting with the metal surface, and form a stable monolayer structure. Raman mapping successfully reveals the spatial boundary migration characteristics of the molecular replacement process, and provides a visual tracking means for the dynamic molecular penetration behavior.

[0075] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A SERS spectroscopic detection method based on molecular substitution, characterized in that, Includes the following steps: A nanocavity structure is constructed on a substrate, the nanocavity structure comprising at least two metal surfaces and at least one metal surface having a molecular layer, with nanoscale gaps formed between the metal surfaces; The matrix is ​​placed in a solution of the target molecules for molecular substitution. After the molecular substitution is completed, SERS spectral detection is performed using a SERS spectral detection device. The molecular layer is polyvinylpyrrolidone; The target molecule solution is methylene blue, benzeneselenool, and biphenyl-4-thiol.

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

3. The SERS spectral detection method according to claim 1, characterized in that, After the first SERS spectral detection, the matrix is ​​placed in the next target molecule solution for molecular replacement, and SERS spectral detection is continued. By continuously replacing the target molecule solution matrix, it can be reused to complete continuous detection or dynamic monitoring.

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