Au@PB@Ag NPs SERS array, preparation method and application thereof

Abstract

The application belongs to the technical field of surface enhanced Raman scattering substrate preparation, and particularly relates to Au@PB@Ag NPs SERS array and a preparation method and application thereof. The method comprises the following steps: preparing monodisperse gold nanoparticles; taking the gold nanoparticles as an inner core, growing a Prussian blue intermediate shell layer on the surface of the particles in situ to obtain Au@PB core-shell composite nanoparticles; depositing a silver nano layer on the outside of the Prussian blue shell layer to obtain Au@PB@Ag three-layer core-shell nanoparticles; performing hydrophilic modification treatment on a solid substrate, dispersing the Au@PB@Ag three-layer core-shell nanoparticles in an aqueous system, forming a continuous film through liquid-liquid interface induced self-assembly, transferring the nanoparticle film to the surface of the hydrophilically modified solid substrate as a whole, and solidifying to obtain the target array. In summary, the application provides a scheme which is simple in preparation process, excellent in process repeatability, and capable of efficiently constructing a large-area and highly ordered arrangement structure.

Description

Technical Field

[0001] This invention belongs to the field of surface-enhanced Raman scattering substrate preparation technology, and particularly relates to an Au@PB@Ag NPsSERS array, its preparation method and application. Background Technology

[0002] Surface-enhanced Raman scattering (SERS) technology, with its core advantages of high sensitivity, rapid response, and non-destructive testing, occupies an important position in the field of biological detection, thus becoming a key technical means for biological sample analysis. As the core support of this technology, the structure and performance of the surface-enhanced Raman scattering active substrate directly determine the detection effect. Currently, the commonly used substrates in the field are mainly plasmonic nanostructures such as metal nanoparticles, nanorods, and nanoarrays.

[0003] The mainstream techniques for preparing surface-enhanced Raman scattering (SERS) substrates currently focus on solution methods and deposition methods, with numerous existing studies exploring different directions within these two approaches. The study "Controllable growth of Au NBPs@Agnanorods modified PDMS as SERS substrate for rapid detection of malachitegreen in aquatic products" uses a controllable growth method to prepare gold nanoparticles with bipyramidal silver coating and modify them with polydimethylsiloxane, but the alignment of the nanoparticles on the substrate is difficult to control precisely. The study "Fabrication of coreshell Au@Ag supraparticles with 3D hotspots via evaporation self-assembly for sensitive surface enhanced Raman scattering detection" prepares core-shell structured gold-coated silver superparticles through evaporation self-assembly, but the uncertainty of liquid film flow during evaporation makes it difficult to guarantee the uniformity of large-area arrays. The study "Template growth of Au / Ag nanocomposites on phosphorene for sensitive SERS detection of pesticides" utilizes a template growth strategy to grow gold and silver nanocomposites on the surface of phosphorene, but the template transfer process easily causes array damage and structural defects. These existing technologies all have common problems that are difficult to avoid in practical applications: (1) The substrate structure is not uniform and the SERS signal reproducibility is poor (RSD is generally >10%) - the prepared substrate generally has a non-uniform structure, which directly leads to the inability to guarantee the repeatability of detection; (2) There is no internal standard correction mechanism, which cannot offset the systematic error - the preparation steps are complicated and the interface assembly is difficult, which affects the molding quality and detection stability; (3) It is difficult to prepare large-area, highly ordered nanoarrays - the uneven distribution of hot spots further reduces the detection sensitivity, which limits the practical application in rapid and highly sensitive detection scenarios.

[0004] Faced with the multiple application limitations brought about by existing technologies, the field of biosensing urgently needs a simple preparation technology that can efficiently complete interface self-assembly, thereby preparing nanoarray substrates with uniform structure, good repeatability, stable signal, and large-area highly ordered arrangement, in order to solve the core pain points in the current preparation and application of surface-enhanced Raman scattering substrates. Summary of the Invention

[0005] The purpose of this invention is to provide an Au@PB@Ag NPs SERS array, its fabrication method, and its application, in order to solve the above-mentioned problems.

[0006] To achieve the above objectives, the present invention provides the following solution: A method for fabricating a self-assembled Au@PB@Ag NPs SERS array, comprising the following steps: Step 1: Prepare monodisperse gold nanoparticles; Step 2: Using the gold nanoparticles as the core, a Prussian blue intermediate shell is grown in situ on the surface of the particles to prepare Au@PB core-shell composite nanoparticles. Step 3: Deposit a silver nanolayer on the outside of the Prussian blue shell of the Au@PB core-shell composite nanoparticles to obtain Au@PB@Ag three-layer core-shell nanoparticles; Step 4: The solid substrate is hydrophilically modified by dispersing Au@PB@Ag three-layer core-shell nanoparticles in an aqueous system and inducing self-assembly at the liquid-liquid interface to form a continuous film with ordered arrangement of nanoparticles at the interface. The ordered assembled nanoparticle film is then transferred to the surface of the hydrophilically modified solid substrate and cured to obtain a self-assembled Au@PB@AgNPs SERS array.

[0007] Preferably, in step 1, the monodisperse gold nanoparticles are prepared by reducing chloroauric acid with trisodium citrate.

[0008] Preferably, in the in-situ growth process of step 2, iron salts and ferrocyanide are used as reaction raw materials, and ascorbic acid is used as a reducing agent.

[0009] Preferably, in the process of depositing the silver nanolayer in step 3, silver nitrate is used as the silver source and ascorbic acid is used as the reducing agent.

[0010] Preferably, in step 4, the solid substrate is a silicon wafer, and the silicon wafer is hydrophilically modified using a piranha solution composed of concentrated sulfuric acid and hydrogen peroxide.

[0011] Preferably, the liquid-liquid interface in step 4 is composed of an aqueous Au@PB@Ag colloid and a mixed organic phase of cyclohexane and ethanol, and the nanoparticles are spontaneously and tightly arranged by interfacial tension.

[0012] An Au@PB@Ag NPs SERS array includes a solid substrate and a monolayer of Au@PB@Ag three-layer core-shell nanoparticles continuously and densely arranged on the surface of the substrate via liquid-liquid interface self-assembly; the nanoparticles have a particle size of 90-100 nm, a particle spacing of 1-5 nm, and the relative standard deviation (RSD) of the SERS signal of the array is less than 5%.

[0013] Preferably, the Au@PB@Ag NPs SERS array is used for rapid detection and non-destructive quantitative analysis of foodborne pathogens.

[0014] Preferably, the foodborne pathogen is Staphylococcus aureus.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: 1. The preparation method is simple and reproducible, and can realize large-area, highly ordered nanoarrays.

[0016] 2. The nanostructure is uniform, and the SERS signal is strong and stable, making it suitable for high-sensitivity detection.

[0017] 3. It is suitable for rapid and non-destructive testing of a variety of biological samples and has good application prospects.

[0018] More importantly, this invention utilizes a dense, ordered array formed through self-assembly at the liquid-liquid interface, which, together with the Prussian blue internal standard layer, produces a significant synergistic enhancement effect. On one hand, the ordered arrangement ensures uniform spacing between nanoparticles and highly consistent distribution of SERS "hot spots," significantly reducing the signal reproducibility (RSD) from 10.47% in the disordered state to 3.38%. On the other hand, the Prussian blue internal standard molecules and the target analyte are in the same electromagnetic enhancement environment, further reducing the signal RSD to 2.71% through ratio correction. The combined enhancement effect far exceeds the simple superposition of a single feature, achieving a simultaneous leap in SERS substrate signal uniformity and detection accuracy, demonstrating the non-obviousness of this invention. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly described below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Figure 1 This is a schematic diagram of the self-assembly process of the present invention; Figure 2 Transmission electron microscope (TEM) image (50 nm) of the gold nanoparticles (AuNPs) prepared in this invention; Figure 3 This is a particle size distribution diagram of the gold nanoparticles (AuNPs) prepared in this invention; Figure 4 Transmission electron microscope (TEM) image (50 nm) of Au@PB core-shell particles prepared in this invention; Figure 5 The particle size distribution diagram of the Au@PB core-shell particles prepared in this invention; Figure 6 Transmission electron microscope (TEM) image (50 nm) of the Au@PB@Ag core-shell particles prepared in this invention; Figure 7 The particle size distribution diagram of the Au@PB@Ag core-shell particles prepared in this invention is shown. Figure 8 The UV-Vis absorption spectra of Au, Au@PB, and Au@PB@Ag particles prepared in this invention are shown. Figure 9 This is a contact angle characterization diagram of the hydrophilic treatment of silicon wafers according to the present invention, wherein, Figure 9 A represents the hydrophobic contact angle diagram of an untreated silicon wafer. Figure 9 B is the hydrophilic contact angle diagram of the silicon wafer after piranha solution treatment; Figure 10 This is a schematic diagram of the self-assembly process of the Au@PB@Ag nanoparticles of the present invention; Figure 11 This is a surface scanning electron microscope (SEM) image (200 nm) of the self-assembled Au@PB@Ag NPs nanoarray of the present invention. Figure 12 This is a magnified optical microscope image (10 μm) of the self-assembled Au@PB@Ag NPs nanoarray of the present invention. Figure 13 This is a high-angle annular dark-field-scanning transmission electron microscope (HAADF-STEM) image and elemental distribution map of the self-assembled Au@PB@Ag NPs nanoarray of this invention. Figure 14 The figures show the SERS performance of Au@PB@Ag nanoparticles before and after self-assembly in this invention. Figure 14 A is a comparison of SERS spectra before and after self-assembly. Figure 14 B is the SERS intensity distribution map before self-assembly (RSD = 10.47%). Figure 14 C represents the SERS intensity distribution after self-assembly (RSD = 3.38%). Figure 15 This is a graph showing the reliability and stability evaluation of Au@PB@Ag nanoparticles before and after self-assembly in this invention. Figure 15 A represents the SERS spectra from the 10 repeated detections prior to self-assembly. Figure 15 B represents the SERS spectra from 10 repeated detections after self-assembly. Figure 15 C is a comparison chart of signal strength and RSD values ​​before and after self-assembly. Figure 15 D represents the long-term stability change of the SERS signal before and after self-assembly; Figure 16This is a simulation analysis diagram of the electromagnetic field of the self-assembled Au@PB@Ag nanoarray using the finite-difference time-domain (FDTD) method. Figure 17 This is a quantitative analysis diagram of the detection of Staphylococcus aureus on the self-assembled Au@PB@Ag substrate of the present invention, wherein, Figure 17 A represents the SERS spectra of Staphylococcus aureus at different concentrations. Figure 17 B represents the standard curve for detecting Staphylococcus aureus concentration.

[0020] Figure 18 The images show transmission electron microscopy (TEM) images and particle size distribution diagrams of traditional Au@Ag core-shell nanoparticles. Figure 18 A is the TEM image of AuNPs. Figure 18 B is the particle size distribution diagram of AuNPs. Figure 18 C is the TEM image of Au@Ag NPs. Figure 18 D represents the particle size distribution of Au@Ag NPs.

[0021] Figure 19 The images show the UV-Vis absorption spectrum and Zeta potential of traditional Au@Ag core-shell nanoparticles. Figure 19 A represents the UV-Vis spectra of AuNPs and Au@Ag NPs. Figure 19 B is the Zeta potential map of Au@Ag NPs. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1

[0024] This embodiment provides a method for preparing a self-assembled SERS tag, which specifically includes the following steps: Step 1: Preparation of AuNPs Take 100 mL of 0.01% (w / v) chloroauric acid solution, heat under reflux until boiling, then quickly add 1 mL of 1% (w / v) trisodium citrate solution, reflux and boil for about 30 min, then stop heating to obtain a wine-red colloid. After the colloid cools naturally to room temperature, scan its UV-Vis spectrum; the maximum absorption wavelength is 531 nm. AuNPs (30±2 nm) should be stored at 4°C protected from light before use.

[0025] Step 2: Preparation of Au@PB 1 mL of 0.1 mol / L ascorbic acid was added to 10 mL of the prepared AuNPs solution. After magnetic stirring for 15 min, 1 mL of 1 mM FeCl3·6H2O and 1 mL of 1 mM K4[Fe(CN)6]·3H2O were added sequentially. After magnetic stirring for 10 min, the solution was centrifuged at 9600 rpm for 5 min. The supernatant was removed, and the solution was redispersed in 10 mL of deionized water to obtain the Au@PB solution.

[0026] Step 3: Preparation of Au@PB@Ag Then add 1 mL of 0.02 g / mL ascorbic acid and 1 mL of 20 mM AgNO3 and continue magnetic stirring for 15 min. Centrifuge at 7600 rpm for 5 min, remove the supernatant, and redisperse in 10 mL of deionized water to obtain Au@PB@Ag solution.

[0027] Step 4: Hydrophilicity Pretreatment of Silicon Wafers The silicon wafers were treated with a piranha solution (a mixture of concentrated sulfuric acid and 30% H2O2 solution in a 7:3 volume ratio). First, the silicon wafers were ultrasonically treated in ethanol for 5 minutes to remove organic matter from the surface and then dried. Then, the silicon wafers were immersed in the piranha solution for 30 minutes to make the surface of the silicon wafers hydrophilic. After immersion, the wafers were washed multiple times with ultrapure water and anhydrous ethanol. The treated hydrophilic silicon wafers were then immersed in ultrapure water for later use.

[0028] Step 5: Liquid-Liquid Interface Self-Assembly and Array Transfer Take 3 mL of Au@PB@Ag colloid and place it in a 5 mL beaker. Use a syringe to slowly add a mixed solution of 1 mL cyclohexane and 1 mL ethanol dropwise from above the liquid surface to induce the nanoparticles to migrate to the liquid-liquid interface. The nanoparticles spontaneously assemble at the interface to form a dense monolayer film. Use tweezers to pick up the pretreated hydrophilic silicon wafer and slowly insert it at a 45° angle below the liquid-liquid interface to transfer the self-assembled Au@PB@Ag NPs nanoarray to the surface of the silicon wafer, thus obtaining the self-assembled Au@PB@Ag NPs SERS array.

[0029] Example 2

[0030] The only difference between this embodiment and Embodiment 1 is that: in the liquid-liquid interface self-assembly process in step five, 4 mL of Au@PB@Ag colloid is placed in a beaker, and a mixed solution of 1 mL of cyclohexane and 1 mL of ethanol is slowly added dropwise from above the liquid surface using a syringe to induce the nanoparticles to migrate to the liquid-liquid interface and spontaneously assemble into a film.

[0031] Example 3

[0032] The only difference between this embodiment and Embodiment 1 is that: in the liquid-liquid interface self-assembly process in step five, 3 mL of Au@PB@Ag colloid is placed in a beaker, and a mixed solution of 1.5 mL of cyclohexane and 0.5 mL of ethanol is slowly added dropwise from above the liquid surface using a syringe to induce the nanoparticles to migrate to the liquid-liquid interface and spontaneously assemble into a film.

[0033] Example 4

[0034] The only difference between this embodiment and Embodiment 1 is that: in the liquid-liquid interface self-assembly process in step five, 3 mL of Au@PB@Ag colloid is placed in a beaker, and a mixed solution of 0.5 mL of cyclohexane and 1.5 mL of ethanol is slowly added dropwise from above the liquid surface using a syringe to induce the nanoparticles to migrate to the liquid-liquid interface and spontaneously assemble into a film.

[0035] Example 5

[0036] The only difference between this embodiment and Embodiment 1 is that n-hexane is used instead of cyclohexane in the liquid-liquid interface self-assembly process in step five. Specifically, 3 mL of Au@PB@Ag colloid is placed in a beaker, and a mixed solution of 1 mL of n-hexane and 1 mL of ethanol is slowly added dropwise from above the liquid surface using a syringe to induce the nanoparticles to migrate to the liquid-liquid interface and spontaneously assemble into a film.

[0037] Example 6

[0038] The only difference between this embodiment and Embodiment 1 is that: before step five, the Au@PB@Ag colloid obtained in step three is first centrifuged and concentrated to increase its original concentration by 1 time. Then, 3 mL of the concentrated Au@PB@Ag colloid is placed in a beaker, and a mixed solution of 1 mL cyclohexane and 1 mL ethanol is slowly added dropwise from above the liquid surface using a syringe to induce the nanoparticles to migrate to the liquid-liquid interface and spontaneously assemble into a film.

[0039] Comparative Example 1: Preparation of a conventional Au@Ag core-shell SERS substrate (1) Preparation of gold nanoparticles Gold nanoparticles were prepared by the trisodium citrate reduction method: 100 mL of 0.01% (w / v) chloroauric acid solution was heated to boiling under reflux, and 1 mL of 1% (w / v) trisodium citrate solution was quickly added. The mixture was then refluxed and boiled for about 30 min to obtain a wine-red colloid. The colloid was cooled to room temperature and stored at 4°C in the dark for later use.

[0040] (2) Preparation of Au@Ag core-shell structure Take 2 mL of the above gold nanoparticle solution, add 10 mL of 0.1 M CTAB and 5 mL of 0.1 M ascorbic acid, and mix well. Add 5 mL of 1 mM AgNO3 solution dropwise while stirring, controlling the dropping rate at 0.5 mL / min. Maintain the reaction temperature at 30℃ and continue the reaction for 2 h. The product is centrifuged and washed three times to remove excess CTAB, and then resuspended in ultrapure water to obtain the conventional Au@Ag core-shell SERS substrate. This substrate does not contain a Prussian blue internal standard layer, and the nanoparticles exist in a disordered colloidal solution form.

[0041] Comparative Example 2: Liquid Au@PB@Ag disordered SERS substrate Au@PB@Ag nanoparticles were prepared according to steps one to three of Example 1. The resulting colloidal solution was not subjected to the hydrophilic modification treatment in step four and the liquid-liquid interface self-assembly operation in step five. Instead, it was directly used as a disordered SERS substrate in colloidal solution form for subsequent comparative testing.

[0042] Experimental Example 1: Nanoparticle Structure Characterization Experiment Experimental objective: To verify the successful construction, particle size uniformity, and optical response characteristics of AuNPs, Au@PB, Au@PB@Ag core-shell structures, and the conventional Au@Ag core-shell structure of Comparative Example 1.

[0043] Experimental methods: The prepared AuNPs, Au@PB, and Au@PB@Ag particles, as well as the conventional Au@Ag core-shell nanoparticles of Comparative Example 1, were subjected to transmission electron microscopy (TEM) morphology observation, particle size distribution statistics, and ultraviolet-visible absorption spectroscopy tests.

[0044] TEM was performed using a JEM-2100 transmission electron microscope from Japan, with an accelerating voltage of 200 kV, a point resolution of 0.23 nm, and a bright-field imaging mode. Sample preparation involved ultrasonically dispersing each particle sample in anhydrous ethanol, then adding the supernatant dropwise onto an ultrathin carbon film copper mesh, drying it under an infrared lamp, and finally observing it.

[0045] Based on the TEM morphology images above, the particle size distribution statistics were performed using ImageJ software to measure the equivalent diameter of at least 200 randomly selected intact particles for each sample, and the average particle size and standard deviation were calculated.

[0046] The UV-Vis absorption spectroscopy measurements were performed using a Hitachi UV-3900H UV-Vis spectrophotometer (Japan), with a wavelength scanning range of 200-800 nm and a scanning speed of 300 nm / min. Baseline calibration was performed before testing using a corresponding blank solvent (deionized water) as a reference.

[0047] Experimental results: AuNPs are uniform spherical nanoparticles with the following morphology: Figure 2 As shown, the particle size is 30±2nm, and the particle size distribution is as follows. Figure 3 As shown; Au@PB particles exhibit a clear core-shell structure, with a morphology as shown. Figure 4 As shown, the particle size distribution is uniform, and the particle size distribution is as follows: Figure 5 As shown; Au@PB@Ag particles form a complete core-shell structure, with a morphology as shown. Figure 6 As shown, the particle size distribution is uniform, and the particle size distribution is as follows: Figure 7 As shown; the characteristic UV absorption peaks of the three particles, Au, Au@PB, and Au@PB@Ag, are 425 nm, 589 nm, and 681 nm, respectively. The UV-Vis absorption spectra are as follows: Figure 8 As shown, the core-shell structure was successfully constructed.

[0048] To further compare the technical effects, a conventional Au@Ag core-shell substrate was also prepared in this experiment (Comparative Example 1). TEM characterization showed that Comparative Example 1 had a clear core-shell structure with an average particle size of approximately 78 nm and a silver shell thickness of approximately 24.5 nm. The morphology and particle size distribution are as follows: Figure 18 As shown. UV-Vis absorption spectroscopy tests indicate that the surface plasmon resonance peak of Comparative Example 1 is located near 450 nm, showing a significant blue shift compared to 525 nm for pure gold nanoparticles. Figure 19 This confirms the uniform coating of the silver shell on the gold core surface and the successful construction of the core-shell structure. Compared with the Au@PB@Ag of this invention, Comparative Example 1 does not contain a Prussian blue intermediate layer, and therefore does not have an internal standard Raman signal.

[0049] Experimental Example 2: Silicon Wafer Hydrophilicity Test Experimental objective: To quantify the effect of hydrophilic modification on silicon wafers and clarify the influence of hydrophilicity on the uniformity of self-assembled arrays.

[0050] Experimental method: First, the silicon wafer was ultrasonically treated in ethanol for 5 min to remove surface organic matter. After drying, the silicon wafer was immersed in a piranha solution of concentrated sulfuric acid and 30% hydrogen peroxide in a 7:3 ratio for 30 min. After immersion, it was repeatedly washed with ultrapure water and anhydrous ethanol to obtain a hydrophilic silicon wafer. The contact angle of the untreated silicon wafer, the silicon wafer treated with piranha solution, and the self-assembled silicon wafer was tested using a contact angle meter.

[0051] The Dataphysics OCA25 optical contact angle meter was used for testing. The test environment was maintained at 25℃ and 50% relative humidity. The test liquid was ultrapure water, and the volume of the single injection droplet was fixed at 25μL. For each type of silicon wafer sample, three samples were randomly selected, and the static contact angle of each sample was measured using a five-point sampling method (center and perimeter). The static contact angle was recorded 5 seconds after the droplet contacted the surface, and the final result was the average of 15 test points.

[0052] Experimental results: like Figure 9 As shown in Figure A, this surface is not conducive to the uniform spreading of nanoparticle aqueous solution. During the transfer process, it is easy to cause liquid film rupture or particle aggregation, which affects the uniformity of the self-assembled array.

[0053] After treatment with piranha solution ( Figure 9 (B) The contact angle on the silicon wafer surface decreased significantly to 7.6°, exhibiting excellent hydrophilic properties. This sharp decrease in contact angle indicates that the strong oxidizing effect of the piranha solution introduced a large number of hydrophilic groups such as hydroxyl groups onto the silicon wafer surface, significantly increasing its surface energy. This highly hydrophilic surface facilitates the uniform spreading of nanoparticle aqueous solutions, providing ideal substrate conditions for the subsequent construction of self-assembled arrays.

[0054] As can be seen, the contact angle of the untreated silicon wafer is 65.5°, which is hydrophobic; after treatment with piranha solution, the contact angle of the silicon wafer drops to 7.6°, which is superhydrophilic; after self-assembly, the contact angle of the silicon wafer rises back to 37.1°, which is still within the hydrophilic range. The hydrophilic modification effect meets the requirements of self-assembly.

[0055] Experimental Example 3: Characterization Experiment of Morphology and Elemental Distribution of Self-Assembled Array Experimental objective: To verify the large-area continuity, tight particle arrangement, and uniform elemental distribution of the self-assembled Au@PB@Ag NPs nanoarray, and to confirm that the array is free of voids, agglomerations, and cracks; and to compare the dispersion state with that of the disordered liquid Au@PB@Ag substrate (Comparative Example 2).

[0056] Experimental methods: The microstructure of the nanoarray surface was observed using scanning electron microscopy (SEM), the macrostructure was observed using optical microscopy, and the elemental distribution of Au, Fe, K, and Ag was analyzed using high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) combined with EDSMapping. Comparative Example 2 (liquid Au@PB@Ag disordered substrate) was directly dropped onto a silicon wafer, dried, and then observed under the same SEM conditions.

[0057] SEM observations were performed using a JSM-7800F field emission scanning electron microscope (SEM) from JEOL (Japan), with an accelerating voltage of 10.0 kV and a working distance of 8 mm. Further, a JEM-2100F field emission transmission electron microscope (SEM) from JEOL (Japan) was used for HAADF-STEM imaging at an accelerating voltage of 200 kV, and area scanning (mapping) analysis was performed on Au, Fe, K, and Ag elements. During area scanning, the pixel dwell time was set to 50 μs, and the total acquisition time was no less than 15 min to ensure high signal-to-noise ratio elemental distribution images.

[0058] Experimental Results: Microscopic morphology analysis revealed that nanoparticles were densely packed on the silicon wafer surface in a monolayer or quasi-monolayer configuration, forming a high-density arrangement. This structure generates numerous hotspot regions, which is crucial for improving SERS performance. The relatively uniform particle size distribution and near-spherical shape ensured the reproducibility of the substrate signal. No obvious voids or multilayer agglomerations were observed within the field of view, indicating that the interfacial tension induced by ethanol at the cyclohexane / water interface effectively drove the self-healing arrangement of the particles, forming an ordered, continuous film. Figure 11 As shown; Macroscopic morphology photographs show that the thin film transferred onto the silicon wafer has a uniform color and is free of cracks or peeling, proving that the liquid-liquid interface assembly method has the ability to prepare centimeter-scale large-area uniform substrates, such as... Figure 12 As shown; EDS elemental mapping analysis further confirmed the high degree of overlap and uniform distribution of Au, Fe, K, and Ag elements throughout the array. The Au signal was concentrated in the particle core, Fe and K, as characteristic elements of Prussian blue, confirmed the existence of the intermediate shell, and Ag was uniformly distributed in the outermost layer. The signal points of each element were extremely uniformly distributed without local agglomeration, verifying the successful construction of the Au@PB@Ag core-shell structure and the high homogeneity of the substrate from a chemical composition perspective. Figure 13 As shown.

[0059] In contrast, SEM observations of Comparative Example 2 (liquid Au@PB@Ag disordered substrate) after drop-drying showed that the nanoparticles were in a random and disordered stacking state, with obvious multilayer agglomeration and void regions in some areas. The interparticle spacing was uneven, and it was impossible to form a large-area continuous dense arrangement structure. This random distribution leads to uneven distribution of "hot spots," which will directly affect the uniformity of the subsequent SERS signal.

[0060] Experiment Example 4: SERS Signal Enhancement and Uniformity Experiment Experimental objective: To verify the SERS signal enhancement and signal uniformity improvement of Au@PB@Ag nanoparticles before and after self-assembly, and to compare them with Comparative Example 1 (traditional Au@Ag core-shell substrate).

[0061] Experimental method: using Prussian blue 2120 cm -1 Characteristic peaks were used as the detection target to perform SERS tests on Au@PB@Ag nanoparticles before and after self-assembly. Comparative Example 1 (traditional Au@Ag core-shell substrate) did not contain a Prussian blue internal standard layer, so the characteristic peaks were used to measure the 4-mercaptobenzoic acid (4-MBA) adsorbed on its silver shell surface at 1078 cm⁻¹. -1The characteristic peaks at the specified locations were the target for detection. A portable Raman spectrometer (laser wavelength: 785 nm, power: 400 mW, integration time: 700 ms) was used to acquire SERS signals. Five different points were collected for each sample, and the average was taken. The repeatability and stability of the probe were simultaneously determined. The signal distribution was analyzed using Raman mapping imaging, and the relative standard deviation (RSD) was calculated.

[0062] Experimental results: Comparing the Raman signals before and after self-assembly reveals that the self-assembled array at 2120 cm⁻¹... -1 The intensity of the Prussian blue characteristic peak at the liquid level is significantly enhanced compared to the liquid state. This enhancement effect is attributed to the close-packed structure formed by self-assembly. When nanoparticles are arranged in an orderly manner at the liquid-liquid interface, high-density electromagnetic field "hot spots" are generated between adjacent particles, which greatly enhances the SERS response of the Prussian blue internal standard molecule located between the gold core and the silver shell. This "sandwich" structure locks the internal standard molecule at the metal interface, placing it precisely in the region of strongest coupling enhancement, thereby achieving effective signal amplification. Figure 14 As shown in A; To assess the signal uniformity of the substrate, Raman imaging analysis was performed on the materials before and after assembly. The results showed that the signal distribution of the substrate before assembly exhibited significant differences in color intensity, with a relative standard deviation (RSD) as high as 10.47%. This indicates that the uneven distribution of "hot spots" caused by random particle dispersion severely limited signal reproducibility. Figure 14 As shown in B; The signal distribution of the self-assembled array is highly uniform in color, and the RSD is significantly reduced to 3.38% (<5%), achieving the excellent reproducibility standard recognized in the SERS field. Figure 14 As shown in C.

[0063] The test results of Comparative Example 1 (traditional Au@Ag core-shell substrate) show that its SERS signal intensity can reach approximately 14000 a.u. (1078 cm⁻¹). -1 However, due to the lack of internal standard molecules for ratio correction, the absolute signal intensity RSD was 12.7% (n=10), and the signal reproducibility was significantly worse than the self-assembled Au@PB@Ag array of this invention (RSD 3.38%). Meanwhile, the traditional Au@Ag core-shell substrate has a lower RSD in the 1800-2800 cm⁻¹ range. -1 The Raman silent region has no characteristic peaks, making it impossible to eliminate systematic errors in the detection process using the internal standard ratio method.

[0064] Test Example 5: Substrate Reliability and Long-Term Stability Test Experimental objective: To verify the detection repeatability and long-term storage stability of the self-assembled Au@PB@Ag NPs SERS array.

[0065] Experimental methods: Ten consecutive random SERS measurements were performed on Au@PB@Ag nanoparticle samples before self-assembly and Au@PB@Ag NPsSERS array samples after self-assembly, and the intensity and repeatability of spectral characteristic peaks were recorded. The samples before and after self-assembly were placed at room temperature for 30 days, and the changes in the SERS signal intensity of the samples were monitored periodically to evaluate the long-term stability of the substrate.

[0066] SERS testing was performed using a portable Raman spectrometer (laser wavelength: 785 nm, power: 400 mW, integration time: 700 ms). For each test, five different regions on the sample surface were randomly selected, and the relative standard deviation (RSD) of the characteristic peak intensities was calculated to assess test repeatability.

[0067] Sample preservation conditions: Samples before and after self-assembly were placed in clean petri dishes, wrapped in aluminum foil to protect them from light, and stored statically in an air environment at room temperature (20~25℃) with relative humidity controlled at 40%~60%. Detection was performed daily for 30 consecutive days, and changes in characteristic peak intensity were recorded to assess the long-term stability of the substrate.

[0068] Experimental results: By comparing the SERS spectra of 10 consecutive random samples before and after self-assembly, it can be found that the 10 spectra of the self-assembled array at 2120 cm⁻¹... -1 The characteristic peaks almost completely overlap in height and have a consistent shape, while the spectrum before assembly shows obvious peak intensity fluctuations. This difference stems from the random Brownian motion of the nanoparticles before assembly, which leads to an inconsistency in the number of particles irradiated by the laser spot and the coupling state. The tightly ordered array formed by self-assembly results in an extremely uniform distribution of surface "hot spots," such as... Figure 15 As shown in A and B; Quantitative analysis further confirmed that the average signal intensity increased from 6053 au to 11844 au after self-assembly, an enhancement of approximately 2 times, and the RSD significantly decreased from 8.51% to 3.75%, reaching the high-quality substrate standard recognized in the SERS field. Figure 15 As shown in C; Long-term stability tests showed that the signal of the unassembled sample dropped sharply within 5-10 days, and was almost undetectable after 15 days. This was mainly attributed to the aggregation or oxidation that easily occurs in the sol system. In contrast, the self-assembled array exhibited excellent storage stability; although the signal decayed slowly over time, it still maintained a signal strength of approximately 10,000 au after 30 days, demonstrating significant detection capability. Figure 15 As shown in D.

[0069] Experimental Example 6: Simulation Experiment of SERS Enhancement Mechanism Experimental objective: To theoretically verify the interparticle "hot spot" enhancement effect and the SERS enhancement mechanism of precise localization of internal standard molecules in the PB layer.

[0070] Experimental method: The local electromagnetic field distribution of a single Au@PB@Ag nanoparticle and a self-assembled dimer structure was numerically simulated using the finite-difference time-domain method (FDTD).

[0071] FDTD simulations were performed using Lumerical FDTD Solutions software. First, a three-dimensional model was established: the Au@PB@Ag nanoparticles were spherical core-shell structures with a total diameter of approximately 92 nm (the Au core diameter, PB layer thickness, and Ag layer thickness were set based on TEM statistical results). A flat silicon substrate was used, with the spheres in direct contact with the substrate. The dimer model consisted of two identical core-shell nanoparticles with a particle spacing of 1 nm. Corresponding material optical constants (dielectric constant and refractive index) were set for each component of the model (Au, PB, Ag). A total field scattering (TFSF) light source and PML boundary conditions were used, and a finely refined grid of 0.5 nm was set at the particle gaps to obtain the local electromagnetic field distribution and enhancement factor.

[0072] Experimental results: Simulation results are as follows Figure 16 As shown, for isolated Au@PB@Ag nanoparticles, the electromagnetic field enhancement mainly occurs on their surface, exhibiting a typical dipole resonance distribution. Although modulated by the core-shell structure, the maximum electric field strength is relatively limited. However, when two nanoparticles self-assemble and closely align to form a dimer structure, extremely strong electromagnetic field aggregation, or "hot spots," appears at the gaps between the particles. This strong enhancement originates from the electromagnetic coupling effect of local surface plasmons between adjacent particles. The region with the maximum field strength precisely covers the core-shell gap where the Prussian blue internal standard molecule is located. This theoretically explains the intrinsic reason for the order-of-magnitude leap in the PB Raman signal after self-assembly. The internal standard molecule is precisely locked in the region with the most intense electromagnetic field enhancement, thereby maximizing signal amplification.

[0073] Experimental Example 7: Quantitative Detection Test of Target Substance Objective: To verify the high sensitivity and quantitative detection capability of the self-assembled Au@PB@Ag NPs SERS array for Staphylococcus aureus.

[0074] Experimental method: Prepare a concentration gradient of 10 2 ~10 8 For Staphylococcus aureus samples with a concentration of CFU / mL, the self-assembled Au@PB@Ag NPs SERS array prepared in this invention was used to perform SERS detection on Staphylococcus aureus samples of different concentrations. A quantitative standard curve was plotted based on the obtained spectral data, and the fitting coefficient of the standard curve was calculated.

[0075] SERS testing was performed using a portable Raman spectrometer (laser wavelength: 785 nm, power: 400 mW, integration time: 700 ms). For each test, five different regions on the sample surface were randomly selected, and the relative standard deviation (RSD) of the characteristic peak intensities was calculated to assess test repeatability.

[0076] Preparation and pretreatment of Staphylococcus aureus samples: Staphylococcus aureus strains were inoculated into LB broth and cultured at 37°C with shaking for 12–16 h until the logarithmic growth phase. After collecting the bacterial suspension, the culture was centrifuged at 5000 rpm for 10 min at 4°C, the supernatant was discarded, and the cells were washed three times with sterile PBS buffer (pH 7.2–7.4) and resuspended in sterile PBS. The concentration of the original bacterial suspension was determined by plate counting, and samples with concentrations ranging from 10⁻⁶ were prepared by serial dilution with PBS. 2 10 3 10 4 10 5 10 6 10 7 10 8 The sample to be tested was prepared at CFU / mL. Take 1 mL of the Staphylococcus aureus bacterial suspension resuspended in PBS and add 10 μL of 10 μM Staphylococcus aureus aptamer (CFU / mL). S. aureus Aptamers (Apt) 5'−SH-GCA ATG GTA CGG TAC TTC CTC GGC ACGTTC TCA GTA GCG CTC GCT GGT CAT CCC ACA GCT ACG TCA AAA GTG CAC GCT ACT TTGCTAA-3') were incubated at 37 ℃ for 30 min to allow the aptamers to fully bind to the bacterial surface. Subsequently, the sample was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the sample was washed twice with PBS to remove unbound aptamers. The sample was then resuspended in PBS to obtain aptamer-labeled Staphylococcus aureus samples. 10 μL of each concentration of aptamer-labeled Staphylococcus aureus sample was added to the surface of a self-assembled Au@PB@AgNPs SERS array and dried in a 37 ℃ oven for 10 min. The interaction between the bacterial surface aptamers and the substrate surface anchored the bacteria to hotspot regions.

[0077] Detection Principle: The detection principle of this invention is based on an aptamer-mediated bacterial anchoring mechanism. First, Staphylococcus aureus is co-incubated with a specific aptamer. The aptamer binds to the bacterial surface with high affinity, forming a "bacteria-aptamer" complex. This complex is then mixed with a self-assembled Au@PB@Ag NPs SERS substrate. The aptamer on the bacterial surface can undergo physical / chemical interactions with specific functional groups on the nano-substrate surface (such as the carboxyl or amino groups exposed on the Ag NPs surface), precisely anchoring the bacteria to SERS hotspot regions. Bacteria enriched in the hotspot regions produce 923 cm⁻¹ of cell wall characteristic components (N-acetylglucosamine in peptidoglycan). -1 Characteristic Raman signal; simultaneously, Prussian blue internal standard molecules in the substrate in the spectral silent region of 2120 cm⁻¹ -1 The stable characteristic peak at that point serves as the calibration internal reference, determined by the ratio I. 923 / I 2120 Ratio normalization correction is performed to eliminate external interference and achieve highly stable and accurate quantitative detection.

[0078] Experimental results: Table 1. Evaluation of SERS signal reproducibility and internal standard correction effect of Staphylococcus aureus (n=8)

[0079] The self-assembled Au@PB@Ag NPs SERS array of this invention was used to detect Staphylococcus aureus. The SERS spectra corresponding to different concentrations of Staphylococcus aureus are shown below. Figure 17 As shown in Figure A, the quantitative standard curve plotted based on the test data is as follows: Figure 17 As shown in Figure B; the linear detection range of this array for Staphylococcus aureus is 10. 2 ~10 8 CFU / mL, the standard curve fitting equation is Y = 0.0677X - 0.0817, and the fitting coefficient R0 is... 2 = 0.9965, indicating that the array has excellent high-sensitivity quantitative detection performance for Staphylococcus aureus.

[0080] Experimental Example 8: Spiked Recovery Test of Complex Matrix Experimental objective: To verify the detection accuracy and resistance to matrix interference of the self-assembled Au@PB@Ag NPs SERS array in the complex biological matrix of fresh camel milk.

[0081] Experimental Methods: Commercially available fresh camel milk was selected as the model matrix. To eliminate the influence of milk fat globules and casein particles on the optical signal, the following pretreatment steps were taken: Fresh camel milk was placed at 4℃ and centrifuged at 8000 r / min for 15 min, and the upper white fat layer was carefully removed; the lower skim milk layer was diluted 10 times with sterile PBS buffer to reduce the matrix viscosity; finally, it was filtered through a 0.22 μm microporous membrane to remove residual large molecular proteins and small impurities, and the resulting clear liquid was used as the matrix for actual sample detection.

[0082] To evaluate the quantitative accuracy of the self-assembled Au@PB@Ag NPs SERS array of this invention in complex biological matrices, a standard spiking method was used for verification. The experiment first involved artificially inoculating a known titer of Staphylococcus aureus suspension into a pretreated blank dairy product matrix to prepare a 10-1... 2 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 8 Spiked samples at four representative concentration gradients of CFU / mL were used, with three independent parallel samples set up for each concentration group to ensure the statistical significance of the data.

[0083] Experimental results: Table 2. Spike recovery rate of Staphylococcus aureus in camel milk samples (n=3)

[0084] Under parallel experimental conditions (n=3), the self-assembled Au@PB@Ag NPs SERS array prepared in this invention achieved a performance of 10 2 ~10 8 The spiked recoveries remained stable between 92.31% and 104.53% across the full CFU / mL range, fully demonstrating that the array still possesses excellent detection accuracy and resistance to matrix interference in complex biological matrices such as fresh camel milk.

[0085] Test Example 9: Comparative Test with Existing Technology Experimental Objective: To systematically compare the performance differences of the self-assembled Au@PB@Ag NPs SERS array (ordered + internal standard) of this invention with those of traditional Au@Ag core-shell substrate (disordered + no internal standard) and liquid Au@PB@Ag disordered substrate (disordered + with internal standard) in terms of signal reproducibility, internal standard correction capability, long-term stability and detection accuracy in complex matrices, and to verify the comprehensive technical advantages of the self-assembled ordered structure combined with the PB internal standard layer of this invention.

[0086] Experimental methods: The self-assembled Au@PB@Ag NPs SERS array prepared in Example 1 was used as the sample of this invention, the conventional Au@Ag core-shell colloidal solution prepared in Example 2 was used as Comparative Example 1, and the liquid Au@PB@Ag colloidal solution prepared in Example 3 was used as Comparative Example 2.

[0087] The SERS testing conditions were standardized as follows: portable Raman spectrometer, laser wavelength 785 nm, power 400 mW, integration time 700 ms. For each detection, spectra were collected from 5 different regions on the sample surface (or in the liquid sample) at random. Each experiment was conducted in parallel 8 times (n=8), and the relative standard deviation (RSD) of the characteristic peak intensity was calculated.

[0088] (1) Signal reproducibility comparison test: using the same concentration (10 6 The detection target was Staphylococcus aureus (CFU / mL). The self-assembled array of this invention uses the I923 / I2120 ratio as the quantitative signal; Comparative Example 1 (conventional Au@Ag) used a 1078 cm-1 ratio. -1 The absolute intensity of the 4-MBA characteristic peak was used as the quantitative signal; for Comparative Example 2 (liquid Au@PB@Ag), the ratio of I923 / I2120 was used as the quantitative signal. The RSD values ​​of the three were calculated respectively.

[0089] (2) Comparison of internal standard correction effect: For Comparative Example 2 (liquid Au@PB@Ag) and the self-assembled array of the present invention, the RSD changes before (absolute intensity) and after (ratio signal) correction of the internal standard were recorded to evaluate the difference in correction efficiency of the internal standard layer in the two states of ordered array and disordered solution. Comparative Example 1 (traditional Au@Ag) does not contain an internal standard layer and can only rely on absolute intensity, so internal standard correction cannot be performed.

[0090] (3) Long-term stability comparison test: The three substrates were placed in a normal temperature (20~25℃), light-proof, and air environment. SERS signals were collected on days 0, 5, 10, 15, 20, 25 and 30, and the trend of characteristic peak intensity change was recorded.

[0091] (4) Comparative test of detection capabilities for complex matrices: using fresh camel milk as the complex biological matrix, at a ratio of 10... 2 10 4 10 6 10 8 Staphylococcus aureus spiked recovery experiments were conducted using four concentration gradients of CFU / mL (n=3) to compare the spiked recovery rates of the three substrates across the entire spiking range.

[0092] Experimental results: (1) Comparison results of signal reproducibility Under the same Staphylococcus aureus detection conditions, the RSD of the signal in Comparative Example 1 (traditional Au@Ag core-shell substrate) was 12.7%, and the RSD of Comparative Example 2 (liquid Au@PB@Ag disordered substrate) was 10.47%. The RSD of the self-assembled Au@PB@Ag NPs SERS array of this invention was only 3.38% (less than 5%). The results indicate that the self-assembled ordered structure ensures uniform spacing between nanoparticles and consistent hotspot distribution, fundamentally solving the problem of large signal fluctuations in traditional disordered substrates.

[0093] (2) Comparison of Internal Standard Correction Effects Although Comparative Example 2 (liquid Au@PB@Ag disordered substrate) contains a PB internal standard layer, the RSD of the absolute signal intensity I~923~ is as high as 12.6% due to the random dispersion of particles. After correction by the I~923~ / I~2120~ ratio, the RSD drops to 10.47%, indicating limited correction effect. The absolute intensity RSD of the self-assembled array of this invention is also about 12%, but after correction by the I~923~ / I~2120~ ratio, the RSD drops sharply to 2.71%. The significant difference in correction effect between the two substrates is attributed to the fact that in the self-assembled array, the internal standard molecules and the target bacteria are in a highly consistent electromagnetic enhancement environment, and their signals "fluctuate synchronously," so the ratio method can effectively offset systematic errors; while in the liquid disordered state, the hot spot environment of the internal standard molecules and the target is significantly different, limiting the correction efficiency. Comparative Example 1 (traditional Au@Ag) does not have an internal standard layer and cannot perform ratio correction, relying only on absolute intensity, with the RSD remaining above 12%.

[0094] (3) Results of long-term stability comparison The signal of Comparative Example 1 (traditional Au@Ag core-shell substrate) experienced a precipitous drop after 5-10 days, and the signal essentially disappeared by day 8. The signal of Comparative Example 2 (liquid Au@PB@Ag disordered substrate) gradually decayed over 5-15 days, and a valid signal was difficult to detect by day 15, mainly due to the aggregation and oxidation of the sol system. The self-assembled Au@PB@Ag NPsSERS array of this invention still maintained more than 80% of the initial signal (approximately 10,000 au) after 30 days. Its excellent stability is attributed to: ① the physical protection of the PB internal standard layer by the gold core-silver shell structure; ② the self-assembled dense array effectively preventing the aggregation and oxidation of nanoparticles.

[0095] (4) Comparison of detection capabilities for complex matrices Comparative Example 1 (traditional Au@Ag core-shell substrate) exhibited severely distorted SERS signals in camel milk matrix due to non-specific adsorption of biomacromolecules such as proteins and fats, as well as light shielding effects, making it impossible to establish an effective quantitative standard curve. Comparative Example 2 (liquid Au@PB@Ag disordered substrate) showed detectable characteristic signals, but due to large signal fluctuations, the recovery rate of low-concentration samples exceeded the acceptable range of 90%–110%. This invention's self-assembled Au@PB@Ag NPs SERS array addresses this issue across the entire spiking concentration range (10... 2 ~10 8 Within the range of CFU / mL, the recovery rate remained stable between 92.31% and 104.53%, fully meeting the requirements for quantitative analysis of complex samples.

[0096] The results of the above four comparative experiments are summarized in Table 3.

[0097] Table 3 Performance Indicators and Evaluation Dimensions Analysis

[0098] Based on the above comparative test results, the self-assembled Au@PB@Ag NPs SERS array of the present invention has the following significant advantages compared with traditional Au@Ag core-shell substrates and liquid Au@PB@Ag disordered substrates: (1) By forming an ordered array through self-assembly at the liquid-liquid interface, the signal reproducibility (RSD) was improved from more than 10% to less than 3%, solving the core problem of uneven signal on the SERS substrate; (2) The synergistic effect of the PB internal standard layer and the ordered array achieved efficient ratio correction, and the RSD was further reduced to 2.71%. Compared with the limited correction effect of the PB internal standard in the disordered state, it proved the irreplaceable nature of the combination of "ordered structure + internal standard layer". (3) The dual effect of core-shell physical protection and dense array structure extends the shelf life of the substrate from less than 15 days to more than 30 days; (4) It maintains excellent quantitative accuracy in complex biological matrices such as fresh camel milk, breaking through the application bottleneck of traditional SERS substrates in real samples where signal distortion and inability to quantify are common.

[0099] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for fabricating a self-assembled Au@PB@Ag NPs SERS array, characterized in that, The steps include: Step 1: Prepare monodisperse gold nanoparticles; Step 2: Using the gold nanoparticles as the core, a Prussian blue intermediate shell is grown in situ on the surface of the particles to prepare Au@PB core-shell composite nanoparticles. Step 3: Deposit a silver nanolayer on the outside of the Prussian blue shell of the Au@PB core-shell composite nanoparticles to obtain Au@PB@Ag three-layer core-shell nanoparticles; Step 4: The solid substrate is hydrophilically modified. Au@PB@Ag three-layer core-shell nanoparticles are dispersed in an aqueous system. Self-assembly is induced through the liquid-liquid interface, so that the nanoparticles are arranged in an orderly manner at the interface to form a continuous film. The ordered assembled nanoparticle film is then transferred to the surface of the hydrophilically modified solid substrate and cured to obtain a self-assembled Au@PB@Ag NPsSERS array.

2. The method for fabricating a self-assembled Au@PB@Ag NPs SERS array according to claim 1, characterized in that: In step 1, the monodisperse gold nanoparticles are prepared by reducing chloroauric acid with trisodium citrate.

3. The method for fabricating a self-assembled Au@PB@Ag NPs SERS array according to claim 1, characterized in that: In the in-situ growth process of step 2, iron salts and ferrocyanides are used as reaction raw materials, and ascorbic acid is used as a reducing agent.

4. The method for fabricating a self-assembled Au@PB@Ag NPs SERS array according to claim 1, characterized in that: In the process of depositing the silver nanolayer in step 3, silver nitrate is used as the silver source and ascorbic acid is used as the reducing agent.

5. The method for fabricating a self-assembled Au@PB@Ag NPs SERS array according to claim 1, characterized in that: In step 4, the solid substrate is a silicon wafer, and the silicon wafer is hydrophilically modified using a piranha solution composed of concentrated sulfuric acid and hydrogen peroxide.

6. The method for fabricating a self-assembled Au@PB@Ag NPs SERS array according to claim 1, characterized in that: In step 4, the liquid-liquid interface is composed of an aqueous Au@PB@Ag colloid and a mixed organic phase of cyclohexane and ethanol, and the nanoparticles are spontaneously and tightly arranged by interfacial tension.

7. An Au@PB@Ag NPs SERS array, characterized in that: It includes a solid substrate and a continuous and densely arranged Au@PB@Ag three-layer core-shell nanoparticle monolayer film on the surface of the substrate through liquid-liquid interface self-assembly; the nanoparticles have a particle size of 90-100 nm, a particle spacing of 1-5 nm, and the relative standard deviation (RSD) of the SERS signal of the array is less than 5%.

8. An application based on the Au@PB@Ag NPs SERS array of claim 7, characterized in that, Application of Au@PB@AgNPs SERS array in rapid detection and non-destructive quantitative analysis of foodborne pathogens.

9. An application based on the Au@PB@Ag NPs SERS array of claim 8, characterized in that, The foodborne pathogen is Staphylococcus aureus.

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