High-density SERS (Surface Enhanced Raman Scattering) substrate and application thereof in detecting active substances of dendrobium

Abstract

The invention discloses a high-density SERS (Surface Enhanced Raman Scattering) substrate and application of the high-density SERS substrate in dendrobium active substance detection, the high-density SERS substrate is formed by self-assembling silver nanoparticles in a water / chloroform / acetone three-phase system, the preparation process of the high-density SERS substrate comprises preparation of the silver nanoparticles and high-density nanostructure assembly, and the obtained high-density SERS substrate is used for SERS measurement in a confocal Raman system. Aiming at the defect of instability of the existing liquid-phase SERS detection, the phase equilibrium principle of a partially mutually soluble three-liquid system is utilized for the first time, a synchronous coupling technology for separating and enriching a high-density hot spot matrix and active ingredients of dendrobium is constructed, and a three-dimensional nanostructure array which is controllably assembled is realized on a three-phase interface; the dispersion uniformity and gap adjustability of the nanoparticles are improved, and hot spots are utilized to the maximum extent; and water-soluble dendrobium active substances are rapidly separated in a three-phase system, so that the water-soluble dendrobium active substances are spontaneously enriched to a hot spot region, and a sensitive and stable Raman signal is obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of ultrasensitive detection of active substances in Dendrobium, and particularly to a high-density SERS substrate and its application in the detection of active substances in Dendrobium. Background Art

[0002] As a highly representative traditional Chinese medicine resource in China, Dendrobium is rich in polysaccharides, flavonoids, alkaloids, and various essential amino acids and trace elements, showing significant pharmacological activities in the fields of immunomodulation, anti-tumor, anti-aging, etc., and having broad application prospects. It is necessary to accelerate the digital innovation of traditional Chinese medicine detection technology, build an intelligent traceability system for the quality of traditional Chinese medicine covering the entire industrial chain, and focus on breaking through the technical bottleneck of rapid detection of active ingredients of genuine medicinal materials.

[0003] That is, by using intelligent detection technologies such as spectral coupling and biosensing, a dynamic monitoring standard for key active ingredients is constructed to address the problems such as the uneven quality of current deep-processed Dendrobium products and the lack of effective control of core ingredients. Therefore, developing a rapid, simple, and efficient analysis method for Dendrobium active substances is of great significance for responding to the strategic deployment of the digital transformation of traditional Chinese medicine, establishing a rapid and accurate detection system for Dendrobium polysaccharides and alkaloids, and promoting the standardization construction of the whole chain of planting - processing - quality control.

[0004] Based on the urgent needs of the development of the Dendrobium industry, the necessity of rapid detection of Dendrobium active substances is mainly reflected in the following aspects: First, traditional detection methods are difficult to meet the on-site detection needs of the Dendrobium industry. The active ingredients of Dendrobium are diverse. Although existing analytical methods such as high-performance liquid chromatography (HPLC) and liquid chromatography - mass spectrometry (LC - MS) have high precision, they usually require cumbersome pretreatment steps, long detection cycles, and high costs, and are difficult to meet the requirements of rapid on-site detection in the industry. Second, surface-enhanced Raman spectroscopy (SERS) technology has the ideal characteristics of rapid detection. SERS has the advantages of ultra-high sensitivity, fingerprint recognition, and few pretreatment steps, and is particularly suitable for the rapid analysis of trace active ingredients in complex matrices. However, the application of SERS in the detection of Dendrobium active ingredients is still in its infancy, and there is still a lack of systematic research on efficient separation and enrichment and stable acquisition strategies. Third, a highly stable SERS substrate is the core of achieving rapid and sensitive detection. By controllably assembling nanomaterials to construct high-density hot spots, the SERS signal can be significantly enhanced. However, the Dendrobium matrix is complex and diverse, and a large number of components such as proteins and polysaccharides may cause unstable aggregation of nanostructures or signal interference. There is an urgent need to develop an SERS substrate with high stability and strong tolerance. Fourth, the complex matrix effect restricts the performance improvement of SERS analysis. Multiple nutrient components in Dendrobium are prone to competitive binding with target molecules or nano-surfaces, significantly reducing the sensitivity and specificity of detection. It is necessary to deeply study the interference mechanism of complex matrices and combine efficient sample pretreatment and target enrichment means to break through the limitation of complex matrix effects on detection signals.

[0005] SERS detection is based on the surface plasmon resonance (SPR) effect generated by noble metal (such as gold, silver, copper, etc.) nanoparticles under specific wavelength excitation, which enhances the Raman signal of target molecules adsorbed on the nanoparticle surface by hundreds of thousands to tens of millions of times. When the gap between nanoparticles is reduced to less than 10 nm, the coupled nanoparticles will generate a strong electromagnetic field, and whenever the target molecule is located in these hot spots, extremely high signal enhancement can be obtained.

[0006] However, in practical applications, SERS faces the balance problem between sensitivity and reproducibility: ① At different positions of the same SERS substrate, the distribution of hot spots may be uneven, resulting in significant differences in Raman signals. ② If the substrate gap is fixed and the spacing is too narrow, the steric hindrance effect will prevent molecules from entering the hot spot area, resulting in most target molecules not being effectively detected. ③ The local electromagnetic field enhancement of SERS rapidly decays with the increase in the distance of the molecule from the metal surface, requiring the target molecule to be as close as possible to or adsorbed on the nanoparticle surface. Therefore, while ensuring the sensitivity and structural uniformity of the active substrate, it is also necessary to fully consider the separation and enrichment of target molecules to enable them to efficiently enter the hot spot area.

[0007] By simultaneously completing the self-assembly of nanoparticles and the enrichment of target molecules at the immiscible liquid / liquid interface (such as oil / water) or partially miscible system, high-gain and high-stability "tunable" liquid-phase SERS detection can be achieved. This liquid-phase SERS strategy uses the interfacial tension between the nanosol and the organic phase to self-organize the particles into a high-density hot spot matrix at the interface, and the target molecules are enriched towards the nanoparticle surface under the drive of solvent extraction or dissolution equilibrium. Compared with the dry state or fixed-gap model, liquid-phase assembly can provide a larger scale, higher density, and more uniform hot spot distribution in three-dimensional space, significantly increasing the effective contact probability between the target molecule and the hot spot. In addition, the liquid-phase detection method also has an extraction and separation process, which can shield or remove complex matrices such as proteins to a certain extent, further improving the sensitivity and specificity of detection.

[0008] However, the liquid-phase SERS strategy still faces the following bottlenecks in the detection of Dendrobium active substances: ① The liquid / liquid or liquid / gas interface is prone to distortion after stirring or oscillation, and the stability of the interfacial assembled nano-hot spot array is difficult to maintain for a long time; ② It is difficult to remove the protein or lipid-soluble coexisting matrix in Dendrobium products in a single step by simple extraction; ③ For water-soluble Dendrobium active substances, how to improve their enrichment efficiency and binding specificity on the nanostructure surface has not been effectively solved. Summary of the Invention

[0009] The purpose of the present invention is to solve the disadvantages existing in the prior art, and to propose a high-density SERS substrate and its application in the detection of Dendrobium active substances.

[0010] To achieve the above object, the present invention adopts the following technical solutions: The present invention first proposes a high-density SERS substrate, which is self-assembled by silver nanoparticles in a water (sol) / chloroform / acetone three-phase system, and its preparation process includes the following steps: S1. Preparation of silver nanoparticles: S101. Synthesis of Ag-NP seed stock solution: 1 mL of an aqueous solution with a mass fraction of 1 wt%, 0.25 mL of an aqueous AgNO3 solution with a mass fraction of 1 wt%, and 0.2 mL of an aqueous NaCl solution with a concentration of 20 mM (i.e., 20 mmol / L) are successively added to 1.05 mL of water under stirring at room temperature. After premixing for 5 min, a citrate-silver-sodium chloride premix is obtained; 80 μL of an aqueous AA solution (ascorbic acid aqueous solution) with a concentration of 0.1 M (i.e., 0.1 mol / L) is added to 47.5 mL of boiling water, and the citrate-silver-sodium chloride premix is immediately added. After heating and stirring for 1 h, it is cooled to room temperature to obtain a brightly colored silver nanoparticle dispersion, namely the Ag-NP seed stock solution; S102. Synthesis of silver nanoparticles (AgNPs): 200 μL of the Ag-NP seed stock solution is added to 4.73 mL of water and stirred at room temperature in a 10 mL glass bottle; subsequently, 70 μL of an aqueous silver-ammonia complex solution with a concentration of 43 mM and 2 mL of an aqueous AA solution with a concentration of 2.5 mM are successively added to the 10 mL glass bottle. After stirring for 1 h, the silver nanoparticles are centrifugally concentrated and then redispersed in an aqueous sodium citrate solution with a mass fraction of 0.02 wt% for storage to obtain a silver nanosol; when the dosage of the silver-ammonia complex is fixed, the size of the AgNPs can be adjusted by changing the dosage of the Ag-NP seeds in the growth solution; S2. High-density nanostructure assembly: 0.2 mL of a chloroform solution containing C 12 with a C

[0011] HDAB concentration of 20 mM (prepared by mixing in a centrifuge tube) and 0.3 mL of acetone are used as the organic phase. Then, 10 μL of the sol droplet is added to the organic phase to form a three-liquid system, and a multilayer nanoparticle structure is assembled at the three-phase interface, that is, a high-density SERS substrate with a nano-shrinkage structure (the gap between Ag nanoparticles is reduced) is obtained. 12 HDAB), and a multilayer nanoparticle structure is assembled at the three-phase interface.

[0012] Preferably, the silver-ammonia complex solution in S102 is prepared by mixing 2 mL of an AgNO3 aqueous solution with a mass fraction of 1 wt% and 800 μL of ammonia water with a mass percentage of 25-28%. The silver-ammonia complex solution is often used as a synthesis for preparing silver nanoparticles by chemical deposition method.

[0013] Preferably, the process of S2 obtains the velocity distribution diagram of the three-phase system at different aggregation times t through COMSOL simulation. The process of the COMSOL simulation is as follows: Use the phase field method (Phase Field) to process the three-phase interface (water / chloroform / acetone); add fluid flow particle tracking to simulate the movement of nanoparticles in the flow field; Material parameter settings, water: density 1000 kg / m 3 , viscosity 1 mPa·s, chloroform: density 1480 kg / m 3 , viscosity 0.54 mPa·s; acetone: density 784 kg / m 3 , viscosity 0.32 mPa·s; silver nanoparticles: density 10490 kg / m 3 , diameter 70 nm (radius 35 nm); Boundary conditions, fluid flow: set the wall to no-slip, and the top / bottom to open boundaries (pressure outlet); particle tracking: initial uniform distribution or release in a specific area; Multiphase flow settings, phase field equation: define the order parameters and interfacial energy of the three phases; gravity effect: enable gravity (9.81 m / s 2 ), and it may sink due to the largest density of chloroform; Particle tracking parameters, Stokes drag force: F drag = (1 / τ p ) × (u fluid - u particle ), where the relaxation time τ p = (2ρ p r 2 ) / (9η fluid ); Brownian force: enable the random diffusion term, diffusion coefficient D = kB×T / (6πηr), assuming the temperature T = 300 K; Boundary conditions, set the fluid interface to "adhesion" to simulate the adsorption behavior of particles at the interface; Solver settings, transient analysis: the time step needs to be less than the minimum dynamic time scale (such as the fluid response time and particle relaxation time); Mesh refinement: refine the mesh in the interface area to improve the accuracy.

[0014] The present invention also proposes the application of the aforementioned high-density SERS substrate in the detection of active substances in Dendrobium, including the following steps: Mix the solution to be detected containing the active substances of Dendrobium and the high-density SERS substrate in a volume ratio of 1:1, and take out the detection droplet with a dropper. Focus the laser spot of the confocal Raman system on the surface of the detection droplet for SERS measurement.

[0015] Preferably, the SERS measurement process is as follows: Collect Raman spectral data on a Horiba confocal Raman system. The collection parameters are: the excitation light wavelength is 633 nm, the laser power is controlled at 0.77 ± 0.03 mW to avoid thermal effects or optical damage caused by the laser. Use a 1800 g / mm high-resolution grating. The laser is focused on the sample through a 50× / 0.75 N.A. objective lens, the spot size is about 1 - 2 μm, the integration time is 1 s, and all the obtained SERS spectra are baseline corrected using LabSpec V6 software.

[0016] Preferably, before detecting the solution to be detected containing the active substances of Dendrobium, the detection sensitivity is explored using crystal violet CV and 4-MBA as probe molecules in advance, that is, mix the detection solution containing CV or 4-MBA and the SERS sol substrate in a volume ratio of 1:1, take out the droplet with a dropper, and focus the laser spot of the confocal Raman system on the surface of the droplet for SERS measurement. The high-density SERS substrate can detect CV concentrations of 10 -9 M - 10 -13 M, can detect CV concentrations of 10 -8 M - 10 -11 M, and a stable dynamic spectrum of the probe SERS signal is collected during the continuous shrinkage of the sol, indicating that the high-density SERS substrate not only has universality for the detection of target substances but also can obtain dynamic spectral signals.

[0017] Preferably, the active substances of Dendrobium include Dendrobium polysaccharide, ferulic acid, kaempferol, apigenin, and gallic acid. The detection limit of the concentration of Dendrobium polysaccharide that the high-density SERS substrate can detect is 10 -2 M, the detection limit of the concentration of ferulic acid is 10 -4 M, and the detection limits of the concentrations of kaempferol, apigenin, and gallic acid are 10 -3 M.

[0018] Preferably, the SERS measurement in S3 can also be used for the quantitative analysis of the active substances of Dendrobium. The steps of quantitative analysis are as follows: Prepare the standard solutions to be detected with the active substances of Dendrobium at concentrations of 10 -5 M - 10 0 M in sequence according to a concentration difference of 5 times, measure the SERS spectra in sequence, and measure the intensity of their characteristic peaks as the standard intensity. For example, the C=O stretching vibration of the carboxylic acid group of ferulic acid is at 1680 - 1700 cm -1For the strong peaks, test each standard intensity in sequence, fit and then plot the RI / C curve showing the change of the standard intensity with concentration; Test the SERS spectrum of the solution to be detected with unknown concentration to obtain the test intensity of the corresponding characteristic peak, and find the concentration corresponding to this intensity in the RI / C curve, which is the estimated test concentration.

[0019] Furthermore, when the SERS spectrum of a certain Dendrobium active substance has multiple characteristic peaks, it is necessary to test the RI / C curves of multiple characteristic peaks. Test the SERS spectrum of the solution to be detected with unknown concentration to obtain the test intensities of each characteristic peak, find the concentrations corresponding to each intensity in the RI / C curve, and take the number-average mean of each concentration, which is the estimated test concentration.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Aiming at the defects of the existing sol substrate for liquid-phase SERS detection, such as the liquid-liquid mixing distortion of water-soluble Dendrobium active substances and the difficulty in maintaining the long-term stability of the nano-hotspot array assembled at the interface, resulting in unstable content detection, the present invention for the first time utilizes the phase equilibrium principle of the partially miscible three-liquid system (sol / chloroform / acetone) to construct a synchronous coupling technology for high-density hotspot matrix and separation and enrichment of Dendrobium active ingredients. By realizing a controllable three-dimensional nanostructure array assembled at the three-phase interface, on the one hand, it improves the dispersion uniformity and gap adjustability of nanoparticles, making the most of the hotspots; on the other hand, it can quickly separate water-soluble Dendrobium active substances in the three-phase system, enabling them to spontaneously enrich to the hotspot area, thereby obtaining sensitive and stable Raman signals; 2. On the basis of the above, the present invention combines signal reading and quantitative analysis methods to establish the dose-effect relationship between the concentration of Dendrobium active ingredients and the SERS signal, and can realize the on-site rapid qualitative and quantitative analysis of Dendrobium active substance samples, providing rapid and accurate detection technical support for the production and sales links of deep-processed Dendrobium products; 3. The present invention can lay an important theoretical foundation and technical guarantee for the quality evaluation and standardization of Dendrobium products, and has important significance for promoting the high-quality development of the Dendrobium industry in China and realizing the digital transformation of traditional Chinese medicine. Description of the Drawings

[0021] Figure 1 It is a set of pictures for the characterization of silver nanoparticles, Figure 1 a is the ultraviolet-visible absorption spectrum of silver nano-sol, Figure 1 b is the SEM image of silver nanoparticles.

[0022] Figure 2 It is a set of pictures for the assembly mechanism of the nanostructure matrix, where Figure 2 a is the phase equilibrium principle diagram of the three-liquid assembled nanostructure, Figure 2b is the velocity distribution diagram of the COMSOL-simulated three-phase system at different aggregation times t. Figure 2 c is the relationship curve between the shrinkage multiple of the nanostructure and the particle gap. Figure 2 d is the SERS signal intensity diagram of different shrinkage multiples of the nanostructure. Figure 2 In d, the SERS spectra of the nanostructures obtained at aggregation times of 0.1 s, 0.06 s, and 0.02 s are shown from top to bottom.

[0023] Figure 3 is the SEM group diagram of the shrunk nanostructures of the silver nanoparticle sol at different magnifications, where Figure 3 a is the SEM image of multiple stacked shrunk nanostructures. Figure 3 b is the SEM image of a single shrunk nanostructure.

[0024] Figure 4 is the data group diagram of the detection of probe molecules by the high-density SERS substrate. In Figure a, the probe molecule is CV. From top to bottom in Figure a, the CV concentrations are 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, and 10 -13 M. In Figure b, the probe molecule is 4-MBA. From top to bottom in Figure b, the 4-MBA concentrations are 10 -8 M, 10 -9 M, 10 -10 M, and 10 -11 M.

[0025] Figure 5 is the data group diagram of the detection of different concentrations of CV by the high-density SERS substrate, where Figure 5 a - e detect CV concentrations of 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, and 10 -13 M.

[0026] Figure 6 is the data group diagram of the detection of different concentrations of 4-MBA by the high-density SERS substrate, where Figure 6 a - d detect 4-MBA concentrations of 10 -8 M, 10 -9 M, 10 -10 M, and 10 -11 M.

[0027] Figure 7 is the spectral diagram of the detection of 10 -2 M dendrobium polysaccharide by the high-density SERS substrate.

[0028] Figure 8 For the detection of 10 -4 M ferulic acid spectrogram.

[0029] Figure 9 For the detection of 10 -3 M kaempferol spectrogram.

[0030] Figure 10 For the detection of 10 -3 M apigenin spectrogram.

[0031] Figure 11 For the detection of 10 -3 M gallic acid spectrogram. Detailed implementation mode

[0032] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the existing well-known technologies. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0033] 1. Preparation and detection of high-density SERS substrate 1.1 Preparation of silver nanoparticles Synthesis of seeds: 1 mL of sodium citrate aqueous solution (1 wt%), 0.25 mL of AgNO3 aqueous solution (1 wt%), and 0.2 mL of NaCl aqueous solution (20 mM, that is, 20 mmol / L) were sequentially added to 1.05 mL of water under stirring at room temperature. After pre-mixing for 5 min, the citrate-silver-sodium chloride premix was quickly added to 47.5 mL of boiling water. Note that 80 μL of ascorbic acid AA aqueous solution (0.1 M, that is, 0.1 mol / L) must be added to the boiling water 1 minute before adding the citrate-silver-sodium chloride premix. After heating and stirring for 1 h, it was cooled to room temperature to obtain a brightly colored silver nanoparticle dispersion.

[0034] Synthesis of silver nanoparticles (AgNPs): A silver-ammonia complex solution was prepared for the synthesis of silver nanoparticles. Generally, 2 mL of AgNO3 aqueous solution (1 wt%) was mixed with 800 μL of ammonia water (25-28%). 200 μL of the Ag-NP seed stock solution was added to water (4.73 mL), and stirred at room temperature in a 10 mL glass bottle. Subsequently, the silver-ammonia complex aqueous solution (70 μL, 43 mM) and the AA aqueous solution (2 mL, 2.5 mM) were sequentially added to the 10 mL glass bottle. After stirring for 1 h, the silver nanoparticles were centrifuged and concentrated, and then redispersed in a 0.02 wt% sodium citrate aqueous solution for storage. When the amount of the silver-ammonia complex was fixed, the size of the AgNPs could be adjusted by changing the amount of Ag-NP seeds in the growth solution.

[0035] 1.2 High-density nanostructure assembly Select water (sol) / chloroform / acetone with two pairs that can be mutually soluble arbitrarily as the nanoparticle assembly environment. Add the cationic surfactant N-dodecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide (C 12 HDAB) to the chloroform phase to obtain a multi-layer nanoparticle structure assembled at the interface.

[0036] 1.3 Steps of detection method 1.3.1 COMSOL simulation Use the phase field method to process the three-phase interface (water / chloroform / acetone); add fluid flow particle tracking to simulate the movement of nanoparticles in the flow field.

[0037] Material parameter settings: water - density 1000 kg / m 3 , viscosity 1 mPa·s; chloroform - density 1480 kg / m 3 , viscosity 0.54 mPa·s; acetone - density 784 kg / m 3 , viscosity 0.32 mPa·s; silver nanoparticles - density 10490 kg / m 3 , diameter 70 nm (radius 35 nm).

[0038] Boundary conditions: for fluid flow, set the wall as no-slip, and the top / bottom as open boundaries (pressure outlets); for particle tracking, set the initial uniform distribution or release in a specific area.

[0039] Multi-phase flow settings: for the phase field equation, define the order parameters and interface energy of the three phases; for the gravity effect, enable gravity (9.81 m / s 2 ), and since chloroform has the largest density, it may sink.

[0040] Particle tracking parameters: Stokes drag force: F drag =(1 / τ p )×(u fluid -u particle ), where the relaxation time τ p =(2ρ p r 2 ) / (9η fluid ); Brownian force: enable the random diffusion term, diffusion coefficient D = kB×T / (6πηr), assuming the temperature T = 300 K.

[0041] Boundary condition: set the fluid interface as "adhesion" to simulate the adsorption behavior of particles at the interface.

[0042] Solver settings, transient analysis: The time step needs to be less than the minimum dynamic time scale (such as the fluid response time and the particle relaxation time). Mesh refinement: Refine the mesh in the interface region to improve the accuracy.

[0043] 1.3.2 Nanoparticle morphology characterization The morphology of the nanoparticles was photographed using an Auriga focused ion beam scanning electron microscope (FIB-SEM). The ultraviolet-visible absorption spectra of the nanomaterials were collected using a UV-2600 spectrophotometer (Shimadzu, Japan).

[0044] 1.4 SERS detection Chloroform (0.2 mL) and 0.3 mL of acetone (mixed with C 12 HDAB) were used as the organic phase, and then 10 μL of the sol droplet was added to the organic phase to form a three-liquid system. The laser spot was focused on the surface of the droplet for SERS measurement.

[0045] 2. Results and discussion Figure 1 Figure shows the ultraviolet-visible absorption spectrum of the AgNPs sol, with a sharp surface plasmon resonance absorption peak at 422 nm, indicating that the nanoparticles have good dispersibility. Through Figure 1 b, it can be seen that the particle size is about 75 nm.

[0046] Figure 2 a is a schematic diagram of the phase equilibrium principle of the silver nanoparticle sol (aqueous phase) / chloroform / acetone three-liquid assembled nanostructure. Figure 2 b shows the velocity distribution of the three-phase system simulated by COMSOL at different times t. The results suggest that the nanoparticles aggregate continuously over time. Figure 2 c shows the change in the particle gap during the continuous aggregation of the nanoparticles. As the nanostructure shrinks continuously, the particle gap decreases continuously. Raman tests were carried out by point scanning and line scanning. As Figure 2 shown in d, the greater the shrinkage multiple of the sol, the greater the SERS signal intensity, indicating that a highly stable nanoparticle gap-tunable nanostructure hot spot matrix can be constructed by the method of three-liquid system assembly.

[0047] Figure 3 a is an assembly diagram of the nanoparticles shown by scanning electron microscopy (SEM). The nanoparticles in the shrunk sol show a large number of aggregated nanostructures. The magnification of the nanoparticles in the shrunk sol was detected. As Figure 3In the shrunk sol shown in b, the spheres are closely packed. The shrunk nanostructure can extend the SERS hotspots to three-dimensional space along the z-axis. Moreover, due to the high density of hotspots on all spatial planes, effective laser interaction can also be achieved. In addition, this structure can adsorb and detect more target molecules in three-dimensional space, enabling highly sensitive sensing applications.

[0048] To further explore the detection sensitivity of the target by the high-density nanoparticle assembly structure, crystal violet CV and 4-MBA were used as probe molecules. Figure 4 a is the SERS spectrum of CV detection. As can be seen from the figure, the characteristics of CV mainly include 1620 cm -1 and 1580 cm -1 attributed to the C-C stretching vibration of the aromatic ring, reflecting the vibration characteristics of the conjugated system; 1375 cm -1 corresponding to the symmetric bending vibration of the methyl group (CH3) in the dimethylamino group; 1175 cm -1 and 1290 cm -1 are the in-plane and out-of-plane bending vibrations of the C-H bond of the aromatic ring respectively; the peak at 915 cm -1 is related to the C-N stretching vibration between the benzene ring and the central nitrogen atom; while 800 cm -1 characterizes the overall breathing vibration mode of the aromatic ring. The intensity of the characteristic peaks of CV decreases with the decrease of the CV concentration. When the CV concentration drops to 10 -13 M, the characteristic peaks at 1620 cm -1 , 1375 cm -1 , 1175 cm -1 are still clearly visible, indicating that the detection limit for CV can reach 10 -13 M.

[0049] Figure 4 b is the SERS spectrum of 4-MBA detection using the prepared high-density substrate. Among them, the strong peak at 1078 cm -1 is attributed to the C-S stretching vibration (Ag-S bond) between the benzene ring and the mercapto group of 4-MBA; 1587 cm -1 and 1484 cm -1 correspond to the C-C conjugated stretching vibration of the benzene ring (attributed to the symmetric and antisymmetric modes respectively); 1141 cm -1 is the in-plane bending vibration of the C-H of the benzene ring; 1185 cm -1 reflects the C-O stretching vibration of the carboxylic acid group (-COOH).

[0050] Figure 5 shows the CV concentrations of 10 -9 M - 10 -13When it is M, the spectra collected during the continuous shrinkage of the sol show that stable SERS signals of CV can be collected during the continuous shrinkage of the nanostructure. The nanomaterial substrate prepared by this method has universality for the detection of target substances, providing a basis for the detection of active substances in Dendrobium officinale.

[0051] Figure 6 The concentrations of 4-MBA are 10 -8 M - 10 -11 M. The spectra collected during the continuous shrinkage of the sol show that stable SERS signals of 4-MBA can be collected during the continuous shrinkage of the nanostructure. The nanomaterial substrate prepared by this method has universality for the detection of target substances. Further, the prepared high-density nanostructure matrix was used as a substrate to detect Dendrobium officinale polysaccharide. As Figure 7 shown, characteristic absorption peaks can be observed, including: CH2 bending vibration near 1450 cm -1 , and aliphatic C-H stretching vibration peaks at 2800 - 3000 cm -1 ; a strong peak of C=O will be shown near 1740 cm -1 , indicating that the SERS substrate prepared by this method can achieve the detection of Dendrobium officinale polysaccharide.

[0052] SERS detection was carried out for the other four active substances in Dendrobium officinale (ferulic acid, kaempferol, apigenin, gallic acid). Among them, ferulic acid shows strong peaks at about 1450 cm -1 (OCH3 deformation vibration), 1650 cm -1 (C=C double bond stretching vibration, acrylic side chain) and 1680 - 1700 cm -1 (carboxylic acid C=O stretching vibration). The vibration of methoxy C-O-C (1150 cm -1 ) marks the structure of o-methoxy-p-hydroxycinnamic acid, as Figure 8 shown.

[0053] The SERS absorption peaks of kaempferol are as Figure 9 shown. The vibration at 1100 - 1150 cm -1 represents the vibration of pyran ring C-O-C, 1280 - 1320 cm -1 (phenolic hydroxyl C-O peak), 1420 cm -1 (CH bending vibration), reflecting the characteristics of flavonol with multiple hydroxyl groups.

[0054] The 1550 - 1580 cm -1 (C ring double bond vibration) of apigenin is a characteristic peak of its flavonoid skeleton. The benzene ring breathing vibration (1600 cm -1 ) and the single hydroxyl C-O peak (1260 - 1300 cm -1 ) reflect the substitution pattern of A / B rings, asFigure 10 as shown

[0055] Due to the structure of gallic acid which is a polyhydroxybenzoic acid, the strong peaks are concentrated at 1680 - 1700 cm -1 (carboxylic acid C=O) and 1600 - 1620 cm -1 (vibration of benzene ring substituted by three hydroxyl groups), and the medium peaks at 1350 - 1420 cm contributed by the hydrogen bond network of hydroxyl groups are as -1 shown Figure 11 as shown

[0056] These four active substances in Dendrobium all share the vibration of the benzene ring as a common feature, and the main source of difference mainly depends on the vibration characteristics of the substituents (the number of carboxylic acid, methoxy, hydroxyl groups and the structure of the flavone ring). The experimental results show that the SERS substrate prepared by this method can realize the detection of phenols, flavonoids and alkaloids in the active substances of Dendrobium, and is expected to provide strong support for the quality control of the Dendrobium industry.

[0057] As mentioned above, only the specific preferred embodiments of the present invention are described, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and all should be covered within the protection scope of the present invention.

Claims

1. A high-density SERS substrate, characterized in that, It is self-assembled from silver nanoparticles in a water / chloroform / acetone three-phase system, and its preparation process includes the following steps: S1. Preparation of silver nanoparticles: S101. Synthesis of Ag-NP seed stock solution: 1 mL of an aqueous solution with a mass fraction of 1 wt%, 0.25 mL of an aqueous AgNO3 solution with a mass fraction of 1 wt%, and 0.2 mL of an aqueous NaCl solution with a concentration of 20 mM are successively added to 1.05 mL of water under stirring at room temperature. After premixing for 5 min, a citrate-silver-sodium chloride premix is obtained; 80 μL of an aqueous AA solution with a concentration of 0.1 M is added to 47.5 mL of boiling water, and the citrate-silver-sodium chloride premix is immediately added. After heating and stirring for 1 h, it is cooled to room temperature to obtain a brightly colored silver nanoparticle dispersion, that is, the Ag-NP seed stock solution; S102. Synthesis of silver nanoparticles: 200 μL of the Ag-NP seed stock solution is added to 4.73 mL of water and stirred at room temperature in a 10 mL glass bottle; subsequently, 70 μL of an aqueous silver-ammonia complex solution with a concentration of 43 mM and 2 mL of an aqueous AA solution with a concentration of 2.5 mM are successively added to the 10 mL glass bottle. After stirring for 1 h, the silver nanoparticles are centrifuged and concentrated, and then redispersed in an aqueous sodium citrate solution with a mass fraction of 0.02 wt% for storage to obtain a silver nano-sol; S2. Assembly of high-density nanostructures: 0.2 mL containing C 12 A chloroform solution with a HDAB concentration of 20 mM and 0.3 mL of acetone are used as the organic phase. Then, 10 μL of sol droplets are added to the organic phase to form a three-liquid system, and a multilayer nanoparticle structure is assembled at the three-phase interface, thus obtaining a high-density SERS substrate with a nano-shrinking structure.

2. The high-density SERS substrate according to claim 1, wherein The silver-ammonia complex solution in S102 is prepared by mixing 2 mL of an aqueous AgNO3 solution with a mass fraction of 1 wt% and 800 μL of ammonia water with a mass percentage of 25-28%. The silver-ammonia complex solution is often used in the synthesis of silver nanoparticles by chemical deposition method.

3. The high-density SERS substrate according to claim 1, wherein The process of S2 is obtained through COMSOL simulation of the velocity distribution map of the three-phase system at different aggregation times t. The process of the COMSOL simulation is as follows: Use the Phase Field phase field method to process the three-phase interface of water / chloroform / acetone; add fluid flow particle tracking to simulate the movement of nanoparticles in the flow field; Material parameter settings, water: density 1000 kg / m 3 , viscosity 1 mPa·s, chloroform: density 1480 kg / m 3 , viscosity 0.54 mPa·s; acetone: density 784 kg / m 3 , viscosity 0.32 mPa·s; silver nanoparticles: density 10490 kg / m 3 , diameter 70 nm; Boundary conditions, fluid flow: set the wall to be non-slip, and the top / bottom to be open boundaries; particle tracking: initial uniform distribution or release in a specific area; Multiphase flow setting, phase field equation: define the order parameters and interfacial energy of the three phases; gravity effect: enable gravity, and since chloroform has the largest density, it may sink; Particle tracking parameter, Stokes drag force: F drag = (1 / τ p ) × (u fluid - u particle ), where the relaxation time τ p = (2ρ p r 2 ) / (9η fluid ); Brownian force: Enable the random diffusion term, diffusion coefficient D = kB × T / (6πηr), assuming temperature T = 300K; Boundary conditions, set the fluid interface to be "adhesive" to simulate the adsorption behavior of particles at the interface; Solver setting, transient analysis: the time step needs to be less than the minimum kinetic time scale; Mesh refinement: refine the mesh in the interface area to improve the accuracy.

4. Use of the high-density SERS substrate according to claims 1-3 in the detection of active substances in Dendrobium, characterized in that, It includes the following steps: Mix the solution to be detected containing the active substances of Dendrobium and the high-density SERS substrate according to a volume ratio of 1:1, take out the detection droplet with a dropper, and focus the laser spot of the confocal Raman system on the surface of the detection droplet for SERS measurement.

5. Use of the high-density SERS substrate according to claim 4 in the detection of active substances in Dendrobium, characterized in that The process of the SERS measurement is as follows: Raman spectral data were collected on a Horiba confocal Raman system with the following acquisition parameters: the excitation light wavelength was 633 nm, the laser power was controlled at 0.77 ± 0.03 mW to avoid thermal effects or optical damage caused by the laser, a 1800 g / mm high-resolution grating was used, the laser was focused on the sample through a 50× / 0.75 N.A. objective lens, the spot size was approximately 1 - 2 μm, the integration time was 1 s, and all the obtained SERS spectra were baseline-corrected using LabSpec V6 software.

6. The application of the high-density SERS substrate according to claim 4 in the detection of active substances in Dendrobium, characterized in that, Before detecting the solution to be detected containing the active substances of Dendrobium officinale, crystal violet CV and 4-MBA are used as probe molecules in advance to explore the detection sensitivity, that is, the detection solution containing CV or 4-MBA and the SERS sol substrate are mixed at a volume ratio of 1:1, and a droplet is taken out with a dropper. The laser spot of the confocal Raman system is focused on the surface of the droplet for SERS measurement. The high-density SERS substrate can detect the CV concentration of 10 -9 M - 10 -13 M, and can detect the CV concentration of 10 -8 M - 10 -11 M.

7. Use of the high-density SERS substrate according to claim 4 in the detection of active substances in Dendrobium, characterized in that The active substances of Dendrobium include polysaccharides, ferulic acid, kaempferol, apigenin, and gallic acid. The detection limit of the concentration of polysaccharides in Dendrobium that the high-density SERS substrate can detect is 10 -2 M, the detection limit of the concentration of ferulic acid is 10 -4 M, and the detection limits of the concentrations of kaempferol, apigenin, and gallic acid are 10 -3 M.

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