SERS (Surface Enhanced Raman Scattering) substrate based on supramolecular plasma gold nanoparticle-mirror surface as well as preparation method and application of SERS substrate

Abstract

The invention relates to the technical field of chiral molecule detection and analysis, in particular to an SERS (Surface Enhanced Raman Scattering) substrate based on a supramolecular plasma gold nanoparticle-mirror surface as well as a preparation method and application of the SERS substrate. The SERS substrate based on the supramolecular plasma gold nanoparticle-mirror surface sequentially comprises a substrate, an AuF thin film layer, an MPBA-beta-CD complex layer and a nanoparticle Au NPs layer from bottom to top, the MPBA-beta-CD complex layer is formed by adsorbing an MPBA-beta-CD complex on the surface of the AuF thin film layer, and a nano gap is formed between the AuF thin film layer and the Au NPs layer. According to the SERS substrate based on the supramolecular plasma gold nanoparticle-mirror surface, beta-CD captures enantiomer molecules to form a supramolecular assembly, nanoscale gap hot spots are accurately manufactured between gold nanoparticles and a gold film, and synchronous recognition and identification of aromatic amino acid enantiomers can be achieved.

Description

Technical Field

[0001] This invention relates to the field of chiral molecular detection and analysis technology, and in particular to a SERS substrate based on supramolecular plasma gold nanoparticles-mirrors, its preparation method, and its application. Background Technology

[0002] Chirality is a fundamental property of nature, decisively influencing the structure and function of biomolecules. Chiral molecules exist as mirror images of each other, called enantiomers. Although enantiomers have similar physicochemical properties, their biological activities, pharmacological effects, and toxicological effects often differ significantly, even to the point of being diametrically opposed. A typical example is the drug thalidomide: its R... The enantiomer has a sedative effect, while S Enantiomers, however, exhibit strong teratogenicity. This difference makes the precise identification and differentiation of chiral molecules crucial in drug development, food safety, disease diagnosis, and life science research. As an important class of bioactive molecules and drug synthesis intermediates, the identification and detection of their different enantiomers is particularly valuable in terms of scientific research and application prospects.

[0003] Existing analytical methods struggle to achieve both high-sensitivity chiral identification and stereostructure characterization in a single detection, often requiring the combined use of multiple techniques. Therefore, developing a novel chiral analysis platform that is rapid, highly sensitive, and provides rich structural information is urgently needed. Surface-enhanced Raman scattering (SERS) technology holds immense potential for chiral identification due to its single-molecule-level sensitivity and fingerprint spectral characteristics. Its signal enhancement originates from localized surface plasmon resonances within noble metal nanostructures, with the size of the inter-nanometer gaps (“hot spots”) being crucial. Smaller gaps (nanometer / subnanometer scale) result in stronger electromagnetic field confinement and more significant signal enhancement. However, the precise control of gap size and morphology on traditional SERS substrates (such as colloidal aggregates or rough films) limits the resolution and reproducibility of chiral analysis.

[0004] In recent years, nanoparticle-mirror (NPoM) structures have attracted attention as a novel type of plasma nanocavity. This structure, by precisely assembling uniform metal nanoparticles onto a smooth metal film, can form sub-nanometer gaps with controllable size and highly localized electromagnetic fields, providing an ideal platform for ultrasensitive detection and structural analysis. Combining NPoM structures with specific chiral recognition capabilities to construct reliable SERS sensing interfaces with high sensitivity and high chiral resolution is a key research direction for promoting the practical application of this technology. However, existing NPoM structure construction methods still face many challenges: in terms of nanoparticle assembly, how to achieve precise, controllable, and scalable assembly of metal nanoparticles on mirror surfaces remains a technical hurdle. Existing NPoM platforms typically use inorganic dielectric layers such as SiO2 and Al2O3 or self-assembled molecular monolayers as gap spacers. While inorganic dielectric layers offer controllable thickness, they lack molecular recognition capabilities; and while self-assembled molecular monolayers can introduce specific functional groups, they suffer from poor structural stability. Therefore, developing an NPoM construction strategy that combines precise gap control, good structural stability, and integrated chirality recognition is of great significance for promoting its practical application in the field of chirality sensing. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a SERS substrate (NPoM / MPBA-) based on supramolecular plasma gold nanoparticles-mirrors. β -CD / SERS), β -CD captures enantiomer molecules to form supramolecular assemblies, precisely creating nanoscale gap hotspots between gold nanoparticles and gold films, enabling simultaneous recognition and identification of aromatic amino acid enantiomers.

[0006] To achieve the above objectives, the present invention provides a SERS substrate based on supramolecular plasma gold nanoparticles-mirrors, wherein the SERS substrate based on supramolecular plasma gold nanoparticles-mirrors comprises, from bottom to top: a substrate, an AuF thin film layer, and an MPBA- β -CD complex layer and Au NPs nanoparticle layer; the MPBA- β -CD complex layer is composed of MPBA- β -CD complexes adsorb onto the surface of the AuF thin film and form nano-gap between the AuF thin film and the Au NPs layer.

[0007] In the substrate of this invention, AuF serves as the optical reflector, and MPBA- β - The CD supramolecular layer has a dual function: firstly, it acts as a precise nanospacer layer, regulating and stabilizing the size of the nanospaces; secondly, its... β-CD cavities can serve as chiral recognition sites, specifically capturing target chiral molecules (such as enantiomers of aromatic amino acids). The added Au NPs act as plasmonic nanoantennas (i.e., nanostructures that capture and enhance the light field through localized surface plasmon resonance). When target molecules are captured in the space composed of Au NPs, AuF, and MPBA- β When the nanocavity gap composed of CD layers is filled, an extremely strong SERS signal can be generated, and its spectral characteristics directly reflect the stereoconfiguration information of the molecule, thereby achieving high-resolution recognition and synchronous characterization of chiral molecules.

[0008] Preferably, the thickness of the AuF thin film layer is 50–200 nm, the particle size of the Au NPs is 60–100 nm, and the substrate is monocrystalline silicon.

[0009] Preferably, the width of the nano-gap is 1–2 nm. MPBA- β -CD complex layers are precisely constructed and tuned to create a gap of 1–2 nm between Au NPs and AuF. This gap acts as a plasma "hot spot" and can generate extremely strong local electromagnetic field enhancement.

[0010] Preferably, the MPBA- β -CD complex composed of MPBA and β -CD is formed by borate ester bonds, and MPBA is adsorbed on the surface of AuF film layer through Au-S bonds, thereby constructing a supramolecular recognition layer on the AuF surface.

[0011] Under the same technical concept, this invention also provides a method for preparing a SERS substrate based on supramolecular plasma gold nanoparticles-mirrors, comprising the following steps: S1. Plasma sputtering of an AuF thin film layer onto the substrate surface; S2. MPBA is incubated and bound on the surface of the AuF thin film layer described in S1 to obtain AuF-MPBA, and the complexation is further carried out. β -CD-based AuF-MPBA- β -CD; S3. Au NPs were prepared by the gold seed growth method in AuF-MPBA- β Au NPs were dropped onto the CD surface and dried to obtain a SERS substrate based on supramolecular plasma gold nanoparticles-mirrors.

[0012] Preferably, the plasma sputtering of the AuF thin film layer on the substrate surface as described in S1 specifically includes: preparing a single-crystal silicon wafer, ultrasonically cleaning it sequentially with acetone, anhydrous ethanol, and ultrapure water, and then drying it; placing the treated single-crystal silicon wafer in the vacuum chamber of the ion sputtering system; and evacuating the chamber to a vacuum level of 1×10⁻⁶. –3Deposition is performed by applying a sputtering voltage of 90–110 V and a sputtering current of 10–40 mA at Pa and below; the deposition time is controlled to be 100–500 seconds to form an AuF thin film with a thickness of 50–200 nm on the surface of a single crystal silicon wafer.

[0013] Preferably, the incubation of the AuF thin film surface with MPBA in S2 specifically includes: immersing the substrate with the sputtered AuF thin film layer described in S1 into an MPBA ethanol solution with a concentration of 0.1–2 mmol / L, and reacting at a temperature of 25–50°C for 4–12 hours; The continued complexation β -CD-based AuF-MPBA- β -CD specifically includes: transferring the substrate to a concentration of 1–10 mmol / L. β In the -CD solution, the solution was prepared using phosphate buffer with a pH of 6.5–10.5 and incubated at 15–30°C for 12–24 hours; after the reaction, the functionalized surface was thoroughly rinsed with ultrapure water and dried under a nitrogen stream to obtain AuF-MPBA- β -CD substrate.

[0014] Preferably, the preparation of Au NPs by the gold seed growth method described in S3 specifically includes: S3.1 Preparation of gold seed solution: Heat 100–150 mL of ultrapure water to 85–110°C and stir for 15–30 minutes. Then add 1–4 mL of sodium citrate solution with a concentration of 20–60 mmol / L and 0.5–2.0 mL of chloroauric acid solution with a concentration of 10–30 mmol / L in sequence. Maintain the temperature and react for 25–35 minutes to obtain gold seed solution. S3.2, First step of growth: Adjust the temperature of the gold seed solution to 80–110℃, add 0.2–0.6 mL of the chloroauric acid solution, and react for 10–30 minutes; repeat this growth step once. S3.3 Second growth step: Mix 30–60 mL of the solution obtained in step S3.2 with 30–60 mL of ultrapure water, heat at 85–110 °C for 15–30 minutes, then add 0.5–2.0 mL of the sodium citrate solution and 0.2–0.6 mL of the chloroauric acid solution sequentially, and react for 10–30 minutes; repeat this second growth step twice to obtain AuNPs with a particle size of 60–100 nm; S3.4 Washing and purification: Centrifuge the obtained Au NPs suspension at 3000–8000 rpm for 5–20 minutes, discard the supernatant, resuspend in ultrapure water and repeat centrifugation and washing 1–3 times. Finally, redisperse the purified Au NPs in ultrapure water to prepare Au NPs suspension, and store at 2–8℃ for later use. The AuF-MPBA- β The specific steps for adding Au NPs to the CD surface include: vertically adding 2–15 μL of the Au NPs suspension prepared in step S3.4 to the AuF-MPBA prepared in step S2. β - The CD substrate surface is left to stand at 15–30°C for 5–15 minutes, and then dried in an oven at 25–35°C to obtain the SERS substrate based on supramolecular plasma gold nanoparticles-mirrors.

[0015] Under the same technical concept, the present invention also provides a SERS substrate based on supramolecular plasma gold nanoparticles-mirrors, which is used for the simultaneous identification and species identification of aromatic amino acid enantiomers.

[0016] The above-described solution of the present invention has the following beneficial effects: (1) This invention utilizes MPBA- β -CD complexes, acting as precise spacers and recognition layers, enabled the construction of size-controllable (1–2 nm) nano-gap structures between gold nanoparticles and gold films, solving the problem of uniform and precise fabrication of "hot spots" in traditional SERS substrates. The constructed NPoM nanocavities confine extremely strong electromagnetic fields to the sub-nanometer scale and combine with... β -CD's specific chiral recognition capability gives the substrate excellent chiral resolution, enabling simultaneous recognition and configuration analysis of chiral molecule enantiomers. (2) The preparation process parameters of the substrate of the present invention are clear and highly controllable, and have good reproducibility and stability, providing a material basis for the development of a reliable and practical chiral SERS sensing platform. The substrate is suitable for the detection of important chiral molecules such as aromatic amino acids, and has broad application prospects in the fields of drug analysis, disease diagnosis and life science research. Attached Figure Description

[0017] Figure 1 The morphological characterization of the AuNPs and NPoM structures in Example 1 is shown. Figure 1 (a) and Figure 1 (b) are SEM images of the synthesized AuNPs at different magnifications. Statistical analysis shows that the average particle size of AuNPs is (76.1 ± 3.5) nm. Figure 1(c) SEM image of the basic NPoM structure.

[0018] Figure 2 This describes the SERS performance characterization of the basic NPoM platform in Example 1. Figure 2 (a) SERS response spectra of the platform for different concentrations of rhodamine 6G (R6G). Figure 2 (b) At an R6G concentration of 10 -6 At time M, measurements were taken at 30 different test points on the substrate surface, and the relative standard deviation (RSD) of the intensity of the key characteristic peaks was analyzed.

[0019] Figure 3 For example, NPoM / MPBA- β - Schematic diagram of the assembly route of the CD sensing platform.

[0020] Figure 4 For example, MPBA and β -UV-Vis titration verification of the formation of a 1:1 host-guest complex of CD in the solution phase. Figure 4 (a) Different MPBA / β UV-Vis absorption spectra at -CD molar ratio. Figure 4 (b) Absorbance at 256 nm as a function of MPBA / β -Titration curve of CD molar ratio change.

[0021] Figure 5 Atomic force microscopy (AFM) characterization of the surface morphology of the functionalized substrate in Example 2. Figure 5 (a) AuF-MPBA- β -CD substrate surface MPBA- β -AFM images and thickness measurements of CD molecular layers. Figure 5 (b) NPoM / MPBA- β - The final AFM topography of the CD structure.

[0022] Figure 6 For example, NPoM / MPBA- β High-resolution transmission electron microscopy (HR-TEM) cross-sectional images of the -CD structure clearly show the MPBA- between AuNPs and AuF. β -Nano gaps formed by CD layers.

[0023] Figure 7 The Raman spectral changes of the substrate during the functionalization process in Example 2 were used to monitor the modification of MPBA and its interaction with... β -CD complexation.

[0024] Figure 8 The water contact angle of the substrate at different functionalization stages in Example 2 is shown below: Figure 8 (a) Bare AuF substrate, Figure 8 (b) AuF-MPBA substrate, Figure 8 (c) AuF-MPBA- β -CD substrate.

[0025] Figure 9 For different substrates (NPoM, NPoM / MPBA, NPoM / MPBA-) in Example 3 β SERS spectra of the enantiomers of tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr) were compared using the -CD method.

[0026] Figure 10 For example, NPoM / MPBA- β -pH optimization results for the chiral recognition performance of the CD platform.

[0027] Figure 11 For example, NPoM / MPBA- β -Optimization results of incubation time for chiral recognition performance on the CD platform.

[0028] Figure 12 For example, NPoM / MPBA is used in Example 3. β -CD platform, characteristic peak of tryptophan (Trp) enantiomer (1529 cm⁻¹) -1 The relative intensity of ) and its enantiomer excess ( ee The linear quantitative relationship between the values.

[0029] Figure 13 To use NPoM / MPBA in Example 3 β -CD platform, characteristic peak of phenylalanine (Phe) enantiomer (998 cm⁻¹) -1 The linear quantitative relationship between the relative intensity of () and its ee value.

[0030] Figure 14 For example, NPoM / MPBA is used in Example 3. β -CD platform, characteristic peak of tyrosine (Tyr) enantiomer (829 cm⁻¹) -1 The linear quantitative relationship between the relative intensity of () and its ee value. Detailed Implementation

[0031] To make the technical problems, solutions, and advantages of this invention clearer, a detailed description will be provided below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0032] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0033] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a locking connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0034] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] Example 1: Construction and performance characterization of the SERS sensing platform: This embodiment details the fabrication process of the basic plasma NPoM structure and its quantitative evaluation of SERS performance. The specific fabrication method consists of the following three steps: The first step involves taking a 0.3 cm × 0.3 cm single-crystal silicon wafer and ultrasonically cleaning it sequentially in acetone, anhydrous ethanol, and ultrapure water for 15 minutes each, followed by drying the surface with high-purity nitrogen. The cleaned single-crystal silicon wafer is then placed on the sample stage of an ion sputtering instrument as a substrate. The vacuum level in the sputtering chamber is evacuated to 5.0 × 10⁻⁶. –4 Pa was used as the sputtering source with a gold target of 99.999% purity. The sputtering operating voltage was set to 105 V, the sputtering current to 30 mA, and the deposition time to 300 seconds, to obtain AuF with a thickness of approximately 100 nm on the surface of a single-crystal silicon wafer.

[0036] 2. Synthesis of AuNPs: Uniformly sized spherical AuNPs were synthesized using a seed-mediated growth method. The specific steps are as follows: (1) Preparation of seed solution: 146 mL of ultrapure water was added to a 250 mL three-necked round-bottom flask, and the flask was heated to 105°C in an oil bath with continuous magnetic stirring (500 rpm). After stabilizing for 20 min, 3.0 mL of a 60 mmol / L sodium citrate aqueous solution was rapidly added. After 5 min, 1.0 mL of a 25 mmol / L chloroauric acid aqueous solution was quickly added. The reaction was maintained at 105°C for 30 min, and the solution color changed from pale yellow to wine red, yielding the gold seed solution.

[0037] (2) First step of growth: Cool the seed solution system to 90°C, add 0.5 mL of the chloroauric acid solution to it at once, and react for 30 minutes. Repeat this step once.

[0038] (3) Second growth step: Take 55 mL of the solution from the first growth step and mix it with 53 mL of ultrapure water preheated to 90 °C in a clean flask. Stabilize at 90 °C for 20 min, then add 1.0 mL of 60 mmol / L sodium citrate solution, followed by 0.5 mL of 25 mmol / L chloroauric acid solution after 5 minutes, and react for 30 minutes. Repeat this growth step twice more to obtain a deep red solution.

[0039] (4) Washing and purification: The resulting deep red solution was centrifuged at 4000 rpm for 10 minutes, the supernatant was discarded, the precipitate was resuspended in ultrapure water and washed by centrifugation again, repeated twice. Finally, the purified AuNPs were dispersed in 5 mL of ultrapure water and stored at 4℃ for later use. Scanning electron microscopy (SEM) images of 100 randomly selected nanoparticles showed that the average particle size of the AuNPs was (76.1 ± 3.5) nm, exhibiting monodisperse spherical shapes. Specific SEM images and particle size distribution are shown below. Figure 1 As shown in (a) and (b).

[0040] 3. NPoM structure assembly: Using a micropipette, 6 μL of the above AuNPs dispersion was vertically added to the central region of the prepared AuF substrate. The sample was allowed to stand at room temperature (25°C) for 10 minutes to allow the nanoparticles to self-assemble under surface tension. The sample was then dried in a 30°C oven for 10 minutes to obtain the basic NPoM structure platform. Specific SEM images are shown below. Figure 1 As shown in (c).

[0041] SERS performance evaluation of the unfunctionalized NPoM platform: Rhodamine 6G (R6G) was used as a model molecule to evaluate the SERS enhancement performance and signal stability of the unfunctionalized NPoM platform. Other conditions were the same as in Example 1, with a series of concentrations ranging from 10... –4 M to 10 –13 An aqueous solution of R6G was used for the NPoM substrate. During testing, 10 μL of a specific concentration of R6G solution was dropped onto the surface of the NPoM substrate, allowed to air dry at room temperature, and then Raman spectroscopy was performed. All SERS spectra were acquired under conditions of 785 nm laser excitation, 5-second integration time, and single accumulation.

[0042] The results are as follows Figure 2 As shown in (a), the platform exhibits extremely high sensitivity to R6G, even at concentrations as low as 10. –13 M can still detect obvious characteristic peaks. According to the formula AEF = (I SERS / I RS ) × (C RS / C SERS ) Calculate and analyze the enhancement factor (AEF), where I SERS and C SERS These represent the SERS intensity and concentration of R6G on the NPoM substrate, respectively, while I RS and C RS These correspond to the conventional Raman intensity and concentration of R6G on an uncoated silicon substrate, respectively. The characteristic peak of R6G is at 1359 cm⁻¹. –1 The strength at that location was calculated, and the AEF was found to be 3.3 × 10⁻⁶. 9 To assess reproducibility, SERS spectra were continuously collected at 30 points at different locations on the substrate surface (R6G concentration: 10). –6 M), the relative standard deviation (RSD) of the critical peak intensity is less than 5.1% ( Figure 2 b). The above data confirms that the NPoM platform has extremely high SERS enhancement capability and excellent signal uniformity.

[0043] Example 2: Assembly and structural characterization of a supramolecular chiral NPoM platform: This embodiment details the supramolecular assembly process of introducing chiral recognition function into the basic NPoM structure, and accurately characterizes the structure using various techniques. Figure 3 Give NPoM / MPBA- β -CD platform synthesis roadmap.

[0044] 1. AuF-MPBA- β -Preparation of CD-functionalized substrates: (1) MPBA modification: The AuF substrate prepared in Example 1 was immersed in 10 mL of 1.0 × 10⁻⁶ MPBA solution. –3The substrate was placed in an ethanol solution of MPBA at 25°C and reacted in the dark for 6 hours. After the reaction, the substrate was removed and thoroughly rinsed with anhydrous ethanol and ultrapure water to remove the physically adsorbed MPBA molecules. Then, it was dried with nitrogen gas to obtain the AuF-MPBA substrate.

[0045] (2) β -CD complexation: The above AuF-MPBA substrate was immersed in 10 mL of PBS prepared with a pH 9.0 concentration of 2.0 × 10⁻⁶. –3 M β Incubate the sample in an aqueous solution of -CD at 25°C for 12 hours. After removal, rinse thoroughly with ultrapure water and dry with nitrogen to obtain AuF-MPBA-. β -CD supramolecular functionalized substrate.

[0046] 2. Solution phase verification during assembly: To verify MPBA and β A 1:1 host-guest complex was formed between -CD, and UV-Vis titration experiments were performed. With the MPBA concentration fixed, different volumes of equal concentrations were gradually added to the complex. β -CD solutions (all dissolved in pH 9.0 PBS). Record the change in absorbance of the mixed solution at 256 nm after each addition. Figure 4 a). Comparing MPBA and absorbance values β Plotting the molar ratio of -CD, the resulting titration curve shows a clear inflection point at a molar ratio of 1:1. Figure 4 (b) confirmed the formation of the 1:1 complex, which provides a theoretical basis for the assembly chemistry of the substrate surface.

[0047] 3. Morphology and dimensional characterization of the functionalized layer and the final NPoM structure: (1) Molecular layer thickness measurement: The AuF surface before and after functionalization was scanned using atomic force microscopy (AFM). The results showed that MPBA- β -The CD supramolecular layer formed a thin film of uniform thickness on the AuF surface, with an average thickness of (1.1 ± 0.2) nm. Figure 5 a).

[0048] (2) Final structural morphology: AuNPs were deposited onto AuF-MPBA- β - On a CD substrate (using the same method as step 3 in Example 1), the final product NPoM / MPBA- is obtained. β -CD. AFM observation shows ( Figure 5 b) AuNPs maintain a uniform distribution on the functionalized substrate surface. The SERS substrate based on supramolecular plasma gold nanoparticles-mirrors consists of, from bottom to top: substrate, AuF thin film layer, MPBA-β -CD complex layer and Au NPs nanoparticle layer; MPBA- β MPBA from the -CD complex layer is adsorbed onto the surface of the AuF film layer via Au-S bonds, forming nano-gap between the AuF film layer and the Au NPs layer.

[0049] (3) Precise measurement of nano-gap: NPoM / MPBA- through focused ion beam technology β -CD structure, cross-sectional samples were prepared and observed using high-resolution transmission electron microscopy. The images clearly show a distinct spacer layer between AuNPs and AuF. Figure 6 The interstitial distance between multiple sites was directly measured, and the statistical average value was (1.4 ± 0.3) nm, which is consistent with the molecular layer thickness measured by AFM.

[0050] 5. Surface chemistry and wettability characterization: Monitoring the functionalization process of the substrate using Raman spectroscopy ( Figure 7 The spectrum of AuF-MPBA shows multiple characteristic peaks of MPBA. β After -CD complexation, the 1574 cm⁻¹ represents the benzene ring b² vibrational mode. –1 The peak was significantly enhanced, especially at 540 cm⁻¹. –1 and 1129 cm –1 The appearance of new characteristic peaks at various locations, attributed to the formation of BOC bonds, directly proves the successful construction of borate ester bonds. Water contact angle testing further corroborates the surface chemical changes: the contact angle of bare AuF is 49.9° ± 1.1°. Figure 8 a), after MPBA modification, it increased to 71.4°±0.7° ( Figure 8 b), and the introduction of hydrophilic elements β After CD, the contact angle slightly decreased to 61.1°±0.4°. Figure 8 c). The above multi-dimensional characterizations collectively confirm NPoM / MPBA- β - Successful construction of the CD chiral SERS platform.

[0051] Example 3 Detection of enantiomers of aromatic amino acids: First, prove NPoM / MPBA- β The -CD / SERS platform possesses the ability to capture chiral molecules and generate highly enhanced SERS signals, and can use tryptophan enantiomers as a model to achieve chiral recognition and enantiomer overload. ee Quantitative analysis of NPoM / MPBA values. β -Verification of the chiral capture and signal enhancement capabilities of the CD platform: The specific operation is as follows: AuF-MPBA-β The -CD substrate was immersed in 0.1 mmol / L L-tryptophan aqueous solution (pH 9.0) and incubated at 25°C for 2 hours. After drying with nitrogen, 6 μL of LAuNPs suspension was added, and the substrate was dried to obtain the detection platform. SERS spectra were acquired using a 785 nm laser (30 mW) with an integration time of 5 seconds. The chiral discrimination performance of the platform was verified as follows: Under the same conditions, detection platforms were prepared using L-tryptophan and D-tryptophan, respectively, and their chiral discrimination at 1529 cm⁻¹ was compared. –1 Differences in SERS spectra of characteristic peaks. To verify quantitative analytical capability, the specific procedure is as follows: Prepare a series of different... ee A mixture of tryptophan enantiomers with a total concentration of 0.1 mmol / L (100% to 100%) was processed as described above and its SERS spectrum was measured at 1529 cm⁻¹. –1 A calibration curve was plotted using peak intensity as the indicator.

[0052] Then, the optimal conditions for the chiral recognition reaction were investigated, and the effects of pH, incubation time, and temperature were verified. The specific procedures were as follows: AuF-MPBA- β -CD substrates were immersed in 0.1 mmol / L L-tryptophan solution (prepared with PBS buffer at pH 7.0–11.0) and incubated at 25°C for 60 minutes to prepare the NPoM platform. SERS measurements were then performed, and the 1529 cm⁻¹ value was recorded. –1 Peak intensity. Incubation time optimization: With pH fixed at 9.0, the effect of incubation time ranging from 15 to 240 minutes was investigated. Only a single variable was changed for each test.

[0053] Finally, to verify NPoM / MPBA- β The CD / SERS platform's universality in recognizing aromatic chiral amino acids continues to target phenylalanine and tyrosine enantiomers. ee The value was then quantitatively analyzed. The specific procedure was the same as described above for tryptophan. ee Quantitative analysis of the values. Special attention was paid to tryptophan at 1529 cm⁻¹. - ¹, Phenylalanine 998 cm –1 And tyrosine 829 cm –1 The intensity of the characteristic peak at that location. Prior to this, the detection conditions were optimized using the controlled variable method.

[0054] Figure 9 For NPoM, NPoM / MPBA and NPoM / MPBA- β -CD substrates identified the SERS spectra of tryptophan, phenylalanine, and tyrosine enantiomers, respectively. The results showed that only NPoM / MPBA- β -CD substrates can achieve enantiomeric differentiation of three aromatic amino acids.

[0055] Figure 10 For NPoM / MPBA- β The results of the investigation into the optimal pH conditions for chiral recognition on the CD platform show that the SERS signal intensity is the highest and the enantiomeric discrimination is the highest at pH 9.0.

[0056] Figure 11 The results of the optimal incubation time optimization show that the signal reaches a stable plateau after 60 minutes of incubation.

[0057] Figure 12 enantiomer of tryptophan ee SERS quantitative analysis results showed a value of 1529 cm. –1 Peak relative intensity and ee The value shows a linear relationship in the range of -100% to 100% (R²=0.997).

[0058] Figure 13 and Figure 14 They are enantiomers of phenylalanine and tyrosine, respectively. ee The SERS quantitative analysis results of the values ​​show that the selected characteristic peak intensity is related to ee The values ​​all showed a good linear relationship (R² were 0.992 and 0.993, respectively).

[0059] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A SERS substrate based on supramolecular plasma gold nanoparticles-mirrors, characterized in that, The SERS substrate based on supramolecular plasma gold nanoparticles-mirrors comprises, from bottom to top: a substrate, an AuF thin film layer, and an MPBA- β -CD complex layer and Au NPs nanoparticle layer; the MPBA- β -CD complex layer is composed of MPBA- β -CD complexes adsorb onto the surface of the AuF thin film and form nano-gap between the AuF thin film and the Au NPs layer.

2. The substrate as claimed in claim 1, characterized in that, The thickness of the AuF thin film is 50–200 nm, the particle size of the Au NPs is 60–100 nm, and the substrate is monocrystalline silicon.

3. The substrate as claimed in claim 1, characterized in that, The width of the nano-gap is 1–2 nm.

4. The substrate as claimed in claim 1, characterized in that, The MPBA- β -CD complex composed of MPBA and β -CD is formed by borate ester bonds, and MPBA is adsorbed on the surface of AuF thin film through Au-S bonds.

5. A method for preparing a SERS substrate based on supramolecular plasma gold nanoparticles-mirrors, characterized in that, Includes the following steps: S1. Plasma sputtering of an AuF thin film layer onto the substrate surface; S2. MPBA is incubated and bound on the surface of the AuF thin film layer described in S1 to obtain AuF-MPBA, and the complexation is further carried out. β -CD-based AuF-MPBA- β -CD; S3. Au NPs were prepared by the gold seed growth method in AuF-MPBA- β Au NPs were dropped onto the CD surface and dried to obtain a SERS substrate based on supramolecular plasma gold nanoparticles-mirrors.

6. The preparation method according to claim 5, characterized in that, The plasma sputtering of an AuF thin film layer on the substrate surface as described in S1 specifically includes: preparing a single-crystal silicon wafer, ultrasonically cleaning it sequentially with acetone, anhydrous ethanol, and ultrapure water, and then drying it; placing the treated single-crystal silicon wafer in the vacuum chamber of the ion sputtering system; and evacuating the chamber to a vacuum level of 1×10⁻⁶. –3 Deposition is performed by applying a sputtering voltage of 90–110 V and a sputtering current of 10–40 mA at Pa and below; the deposition time is controlled to be 100–500 seconds to form an AuF thin film with a thickness of 50–200 nm on the surface of a single crystal silicon wafer.

7. The preparation method according to claim 5, characterized in that, The specific steps of S2 for incubating the AuF thin film surface with MPBA include: immersing the substrate with the sputtered AuF thin film layer described in S1 into an MPBA ethanol solution with a concentration of 0.1–2 mmol / L, and reacting at a temperature of 25–50°C for 4–12 hours. The continued complexation β -CD-based AuF-MPBA- β -CD specifically includes: transferring the substrate to a concentration of 1–10 mmol / L. β In the -CD solution, the solution was prepared using phosphate buffer with a pH of 6.5–10.5 and incubated at 15–30°C for 12–24 hours; after the reaction, the functionalized surface was rinsed with ultrapure water and dried under a nitrogen stream to obtain AuF-MPBA- β -CD substrate.

8. The preparation method according to claim 5, characterized in that, The preparation of Au NPs by the gold seed growth method described in S3 specifically includes: S3.1 Preparation of gold seed solution: Heat 100–150 mL of ultrapure water to 85–110 °C and stir for 15–30 minutes. Then add 1–4 mL of sodium citrate solution with a concentration of 20–60 mmol / L and 0.5–2.0 mL of chloroauric acid solution with a concentration of 10–30 mmol / L in sequence. Maintain the temperature and react for 25–35 minutes to obtain gold seed solution. S3.2, First step of growth: Adjust the temperature of the gold seed solution to 80–110℃, add 0.2–0.6 mL of the chloroauric acid solution, and react for 10–30 minutes; repeat this growth step once. S3.3 Second growth step: Mix 30–60 mL of the solution obtained in step S3.2 with 30–60 mL of ultrapure water, heat at 85–110 °C for 15–30 minutes, then add 0.5–2.0 mL of sodium citrate solution and 0.2–0.6 mL of chloroauric acid solution sequentially, and react for 10–30 minutes; repeat this second growth step twice to obtain Au NPs with a particle size of 60–100 nm; S3.4 Washing and purification: Centrifuge the obtained Au NPs suspension at 3000–8000 rpm for 5–20 minutes, discard the supernatant, resuspend in ultrapure water and repeat centrifugation and washing 1–3 times. Finally, redisperse the purified Au NPs in ultrapure water to prepare Au NPs suspension, and store at 2–8℃ for later use. The AuF-MPBA- β The specific steps for adding Au NPs to the CD surface include: vertically adding 2–15 μL of the Au NPs suspension prepared in step S3.4 to the AuF-MPBA prepared in step S2. β - The CD substrate surface is left to stand at 15–30°C for 5–15 minutes, and then dried in an oven at 25–35°C to obtain the SERS substrate based on supramolecular plasma gold nanoparticles-mirrors.

9. A SERS substrate based on supramolecular plasma gold nanoparticles-mirrors as described in any one of claims 1-4, or a SERS substrate based on supramolecular plasma gold nanoparticles-mirrors prepared by the preparation method described in any one of claims 5-8, characterized in that, The SERS substrate based on supramolecular plasma gold nanoparticles-mirrors is used for the simultaneous identification and species identification of aromatic amino acid enantiomers.

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