A gap-adjustable chiral threaded gold nanorod@zif-8-nh2 gold film SERS substrate, a preparation method and application thereof

Abstract

This invention relates to the field of chiral molecular detection and analysis technology, protecting a chiral threaded gold nanorod@ZIF-8-NH2 gold film SERS substrate with adjustable gap, its preparation method, and its applications. The SERS substrate comprises, from bottom to top, a substrate, an AuF film layer, a chiral threaded gold nanorod core, and a chiral threaded gold nanorod@ZIF-8-NH2 shell layer covering its surface—a chiral threaded gold nanorod@ZIF-8-NH2 composite structure layer. The ZIF-8-NH2 shell layer forms nano-gap between the chiral threaded gold nanorod and the AuF film layer. The size of the nano-gap can be adjusted by controlling the thickness of the shell layer. Furthermore, utilizing the porous enrichment effect of ZIF-8-NH2 and the multiple interactions between the surface NH2 groups and chiral amino acids, combined with the intrinsic chiral optical activity of the chiral threaded gold nanorod, highly sensitive, high-resolution, and universally applicable recognition and identification of multiple (at least seven or more) chiral 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 chiral threaded gold nanorod@ZIF-8-NH2 gold film SERS substrate with adjustable gap, its preparation method and application. Background Technology

[0002] Chiral small molecule metabolites, due to their stereospecificity, possess unique advantages in regulating physiological processes and reflecting the body's health status. Amino acids, as core chiral metabolites, show that abnormal D / L enantiomer ratios are closely related to the pathological progression of various major diseases. It has been reported that the levels of D-alanine (D-Ala) and D-proline (D-Pro) are significantly elevated in gastric cancer patients; D-serine (D-Ser), a key endogenous ligand for the NMDA receptor, is directly associated with concentration imbalances in schizophrenia (decreased concentration), Alzheimer's disease (increased concentration), brain injury, epilepsy, and amyotrophic lateral sclerosis (ALS). Furthermore, L-lysine (L-Lys) levels show significant changes in patients with acute myeloid leukemia. Therefore, amino acid chirality analysis is of great significance for early disease diagnosis and prognosis.

[0003] Existing chiral amino acid detection technologies suffer from low sensitivity and throughput, high cost, and long processing times (ranging from hours to days), making it difficult to meet the needs of rapid clinical diagnosis. The current gold standard for chiral amino acid molecular detection is chiral liquid / gas chromatography-mass spectrometry (LC / GC-MS), which, while offering high sensitivity (picomolar levels), still suffers from limitations such as expensive equipment and the need for specialized technicians. Furthermore, chiral derivatization reagents are required to convert amino acids into diastereomers, a cumbersome process that easily introduces chiral impurities. Surface-enhanced Raman spectroscopy (SERS), with its molecular fingerprint specificity and single-molecule detection potential, has become an ideal tool for trace metabolite analysis. However, applying SERS to achieve highly sensitive detection of chiral amino acids still faces two unresolved issues. First, plasmon resonance substrates are generally achiral and cannot directly distinguish enantiomers, requiring the artificial introduction of a chiral environment. Second, it is difficult to confine the analyte to the hotspot region of the SERS substrate, resulting in low detection sensitivity and resolution.

[0004] In recent years, researchers have attempted to endow SERS substrates with chiral recognition capabilities through chiral ligand modification or chiral recognition elements. However, since the design of chiral recognition interfaces often relies on specific intermolecular interactions (such as hydrogen bonding, hydrophobicity, and inclusion interactions), it is difficult to achieve universal recognition of chiral molecules with different structures. Recently, by constructing plasmon nanostructures with intrinsic chiral optical activity, chirality has been directly transferred from the molecular scale to the nanoscale, opening a new path for the development of universal chiral SERS sensing platforms. On the other hand, to achieve sensitive detection of trace chiral molecules, SERS substrates need to possess extremely high signal enhancement capabilities. Nanoparticle-mirror (NPoM) structures, by constructing sub-nanometer gaps between nanoparticles and metal mirrors, can generate extremely localized electromagnetic hotspots, thereby achieving significant SERS signal enhancement. However, while the commonly used self-assembled monolayer strategy can construct nanoscale gaps, it is difficult to guarantee the high uniformity of the gap size. Inherent defects in the molecular layer, tilting of the molecular chain, or conformational changes can all lead to fluctuations in the gap width, thereby impairing the reproducibility and reliability of SERS signals. For example, patent publication number CN121656221A discloses an enantioselective surface-enhanced Raman scattering substrate that uses chiral molecules to induce the synthesis of chiral helical gold nanorods (L-HAuNRs). By adjusting the solution pH and adding thiol ligands, the self-assembly of the chiral gold nanorods is promoted, and then they are coupled with a deposited gold nanofilm (AuF). More importantly, once the NPoM structure formed by this method is finalized, its gap distance is fixed. This severely limits its application potential in cutting-edge fields such as dynamic sensing and reconfigurable detection. Summary of the Invention

[0005] This invention provides a chiral threaded gold nanorod@ZIF-8-NH2 gold film SERS substrate with adjustable gap, its preparation method and application, with the aim of solving the above-mentioned problems existing in the background art.

[0006] To achieve the above objectives, embodiments of the present invention provide a chiral threaded gold nanorod@ZIF-8-NH2 gold film SERS substrate with adjustable gap, its preparation method, and its application. By adjusting the thickness of the ZIF-8-NH2 shell, the nano-gap size can be precisely adjusted. By utilizing the porous enrichment effect of ZIF-8-NH2 and the multiple interactions between the surface NH2 groups and chiral amino acids, combined with the intrinsic chiral optical activity of the chiral threaded gold nanorod, it is possible to achieve highly sensitive, high-resolution, and universal recognition and identification of multiple (at least 7 or more) chiral amino acid enantiomers.

[0007] One aspect of the present invention provides a chiral threaded gold nanorods@ZIF-8-NH2 gold film SERS substrate with adjustable gap, comprising, from bottom to top, a substrate, an AuF film layer, and a chiral threaded gold nanorods@ZIF-8-NH2 composite structure layer; The chiral threaded gold nanorod@ZIF-8-NH2 composite structure layer includes a chiral threaded gold nanorod core and a ZIF-8-NH2 shell coating on its surface; The ZIF-8-NH2 shell forms a uniform nanogap between the chiral threaded gold nanorods and the AuF film. The size of the nanogap can be adjusted by controlling the thickness of the ZIF-8-NH2 shell. The ZIF-8-NH2 shell also enriches the target chiral molecules and provides amino active groups to enhance the interaction with chiral amino acids.

[0008] In the substrate of this invention, the AuF film serves as an optical reflector, forming an NPoM plasmonic nanocavity together with chiral threaded gold nanorods. The chiral threaded gold nanorods possess intrinsic chiral optical activity, enabling them to directly couple with chiral amino acids of different configurations in a differentiated plasmon resonance manner, thereby achieving universal chiral recognition. The ZIF-8-NH2 shell coating the surface of the chiral threaded gold nanorods has three functions: first, it acts as a precise and tunable nanospacer layer, controlling the shell thickness to regulate the size of the nanospace and thus optimize the electromagnetic field enhancement intensity of the plasmon hotspots; second, it utilizes the porous structure of ZIF-8 to enrich target chiral molecules, increasing the local concentration; and third, the abundant amino groups on its surface can specifically bind to chiral amino acids through multiple interactions such as hydrogen bonding and electrostatic interactions, enhancing the enantiomeric resolution. When target molecules are enriched and trapped in the intercellular space of a nanocavity composed of chiral threaded gold nanorods, AuF and ZIF-8-NH2 shells, an extremely strong SERS signal can be generated, and the spectral characteristics can directly reflect the stereoconfiguration information of the molecules, thereby achieving highly sensitive, high-resolution and universal detection of chiral amino acids.

[0009] Preferably, the thickness of the AuF film is 50~200 nm; the substrate is monocrystalline silicon. Preferably, the thickness of the ZIF-8-NH2 shell is 3~30 nm.

[0010] Preferably, the surface of the chiral threaded gold nanorod has a threaded chiral structure; wherein the chiral structure is an R configuration or an S configuration; the length of the chiral threaded gold nanorod is 120~200 nm and the diameter is 40~100 nm.

[0011] An embodiment of the present invention also provides a method for preparing the above-mentioned chiral threaded gold nanorods@ZIF-8-NH2 gold film SERS substrate with adjustable gap, comprising the following steps: S1: AuF film is deposited by plasma sputtering on a pretreated, clean substrate. S2: Chiral spiral gold nanorods were obtained by surface modification with chiral inducing agents. S3: The chiral threaded gold nanorods are dispersed in a Zn(NO3)2 reaction system containing 2-methylimidazole and 2-aminobenzimidazole, and ZIF-8-NH2 is coated on the surface of the chiral threaded gold nanorods by coordination self-assembly to obtain the chiral threaded gold nanorod@ZIF-8-NH2 complex. S4: Assemble the chiral threaded gold nanorods@ZIF-8-NH2 composite onto the surface of the AuF film, and dry it to obtain the chiral threaded gold nanorods@ZIF-8-NH2 gold film SERS substrate with adjustable gap.

[0012] Preferably, in step S1, the pretreatment includes: sequential ultrasonic cleaning with acetone, anhydrous ethanol, and ultrapure water, followed by drying; the sputtering deposition process involves a vacuum level below 1×10⁻⁶. -3 Pa, sputtering voltage of 90~110 V, sputtering current of 10~40 mA, and deposition time of 100~500 seconds.

[0013] Preferably, in step S2, the preparation process of the chiral threaded gold nanorods specifically includes: S2.1: Under stirring at 500 rpm, mix 4-6 mL of 0.4-0.6 mM chloroauric acid solution with 4-6 mL of 0.15-0.25 M hexadecyltrimethylammonium bromide (CTAB) solution, then add 0.5-0.7 mL of 8-12 mM sodium borohydride ice water solution, and inject under vigorous stirring at 1200 rpm for 1-3 minutes. The resulting seed solution is aged in a water bath at 25-35℃ for 1-3 hours. S2.2: Dissolve 1.3–1.5 g of cetyltrimethylammonium bromide and 0.20–0.30 g of sodium oleate in 45–55 mL of ultrapure water, and stir in a water bath at 45–55 °C until completely dissolved; cool to 25–35 °C, add 3.0–4.0 mL of 3–5 mM silver nitrate solution, let stand for 10–20 minutes, then add 45–55 mL of 0.8–1.2 mM chloroauric acid solution, and stir for 80–100 minutes until the solution becomes colorless; add 0.5–0.7 mL of 37% hydrochloric acid, and stir at 350–450 rpm for 10–20 minutes, then add 1.0–1.5 mL of 0.06–0.07 M ascorbic acid, and stir at 650–750 rpm for 25–35 seconds; finally add 0.06–0.10 g of sodium oleate solution. The seed solution obtained in step S2.1 was stirred for 25-35 seconds and then allowed to stand at 25-35°C for 10-15 hours; the product was collected by centrifugation at 6500-7500 rpm for 8-12 minutes. S2.3: Dissolve 30-40 mg of chiral inducer and 1.5-1.7 g of hexadecyltrimethylammonium chloride (CTAC) in 45-55 mL of water, heat and stir at 55-65°C until completely dissolved, cool to room temperature to obtain a chiral inducer / hexadecyltrimethylammonium chloride solution, wherein the concentration of hexadecyltrimethylammonium chloride is 90-110 mM and the concentration of chiral inducer is 2.0-3.0 mM; wash the gold nanorods obtained in step S2.2 at least twice with 8-12 mL of 8-12 mM hexadecyltrimethylammonium chloride solution, then wash twice with the above chiral inducer / hexadecyltrimethylammonium chloride solution, and finally redisperse them in the solution and age overnight; mix 20-25 mL of ultrapure water with 5-7 mL of the above chiral inducer / hexadecyltrimethylammonium chloride solution, add 250-350 µL of 40-60 mM tetrachloroauric acid solution, and stir at 400-600... After stirring at rpm for 25-35 seconds, let stand at room temperature for 4-6 minutes; then add 250-350 µL of the gold nanorod colloid obtained in step S2.2, and quickly inject 550-650 µL of 0.7-0.9 M ascorbic acid. After vigorous shaking, let stand for 15-25 minutes; after the reaction is complete, centrifuge at 2500-3500 rpm for 8-12 minutes, repeat the centrifugation twice, and redisperse the precipitate in 0.8-1.2 mL of 8-12 mM hexadecyltrimethylammonium chloride solution to obtain chiral spiral gold nanorods, which are stored at 2-8℃ for later use. The chiral inducer is R-(+)-1,1'-bi-2-naphthylamine or S-(-)-1,1'-bi-2-naphthylamine.

[0014] Preferably, step S3 specifically includes: washing the chiral threaded gold nanorods after centrifuging at 2500-3500 rpm for 8-12 minutes, and immediately redispersing them with ultrapure water to obtain a chiral threaded gold nanorod dispersion; adding 8-12 mL of the chiral threaded gold nanorod dispersion and 0-1000 µL of a 0.5-5 mM hexadecyltrimethylammonium bromide (CTAB) solution sequentially to a reaction vessel, mixing thoroughly, and then adding 8-12 mL of a mixed ligand solution containing 0.8-1.0 M 2-methylimidazole and 1%-50% by mass of 2-aminobenzimidazole, stirring at 350-450 rpm for 4-6 minutes; then adding 8-12 mL of... A Zn(NO3)2 aqueous solution was stirred for 4–6 minutes; the mixture was then allowed to stand for 60–120 minutes to prepare chiral spiral gold nanorods@ZIF-8-NH2 composites. Methanol was then added, and the mixture was centrifuged at 3000–4000 rpm for 4–6 minutes. The mixture was washed twice, reconstituted in methanol, and stored at 2–8°C for later use. The thickness of the ZIF-8-NH2 shell was controlled to be 5–30 nm by adjusting the amounts of CTAB and 2-aminobenzimidazole. Preferably, the amount of 2-aminobenzimidazole was 1%, 3%, …, 10%, 20%, 30%, …, 50% of the mass of 2-methylimidazole. The amount of hexadecyltrimethylammonium bromide (CTAB) solution was 0 µL, 50 µL, …, 180 µL, …, 360 µL, …, 720 µL, …, 1000 µL. More preferably, the amount of 2-aminobenzimidazole was 10% of the mass of 2-methylimidazole.

[0015] Preferably, in step S4, the chiral threaded gold nanorods@ZIF-8-NH2 composite is dispersed in ultrapure water to obtain a composite suspension; 2~15 μL of the composite suspension is vertically dropped onto the surface of the AuF membrane, allowed to stand at 15~30℃ for 5~15 minutes, and then dried at 25~35℃ to obtain the chiral threaded gold nanorods@ZIF-8-NH2 gold film SERS substrate with adjustable gap.

[0016] Another aspect of the present invention provides the application of the above-described chiral threaded gold nanorods@ZIF-8-NH2 gold film SERS substrate with adjustable gap or the chiral threaded gold nanorods@ZIF-8-NH2 gold film SERS substrate with adjustable gap prepared by the above-described preparation method in the field of non-aromatic amino acid molecule detection, wherein the non-aromatic amino acid molecule is at least one of lysine, isoleucine, asparagine, serine, proline, leucine and alanine.

[0017] The above-described solution of the present invention has the following beneficial effects: (1) This invention uses a ZIF-8-NH2 shell as a nano-spacer layer with precisely adjustable thickness to construct a nano-gap with adjustable size (5–30 nm) between chiral threaded gold nanorods and AuF film, solving the problem that the gap size is fixed and cannot be dynamically adjusted in the traditional self-assembled monolayer method; the constructed NPoM nanocavity can realize the on-demand optimization of plasmon hotspots, and combined with the intrinsic chiral optical activity of chiral threaded gold nanorods, the substrate has universal chiral resolution capability, and can realize high-sensitivity, high-resolution simultaneous recognition and configuration analysis of at least 7 chiral amino acid enantiomers; (2) In this invention, the ZIF-8-NH2 shell has the dual functions of molecular enrichment and active group enhancement: its porous structure can effectively enrich the target chiral molecules and increase the local concentration; the abundant NH2 groups on the surface strengthen the specific binding with chiral amino acids through multiple interactions such as hydrogen bonding and electrostatics, further improving the detection sensitivity and enantioselectivity. (3) The preparation process parameters of the substrate of the present invention are clear and highly controllable. The gap size can be easily controlled by adjusting the MOF synthesis conditions to enhance the chiral SERS recognition, and it has good reproducibility and stability. The substrate is suitable for the detection of a variety of chiral amino acids and can recognize a variety of amino acids from aromatic to aliphatic and polar amino acids with low Raman cross sections. It has broad application prospects in the fields of drug analysis, disease diagnosis, food safety and life science research. Attached Figure Description

[0018] 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 introduced 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.

[0019] Figure 1 These are scanning electron microscope (SEM) images of gold nanorods, R-configuration and S-configuration chiral spiral gold nanorods in embodiments of the present invention; wherein, Figure 1 A represents gold nanorods. Figure 1 B represents an R-configuration chiral spiral gold nanorod. Figure 1 C represents an S-configuration chiral threaded gold nanorod.

[0020] Figure 2 This is a statistical distribution diagram of the particle size of R-configuration and S-configuration chiral threaded gold nanorods in an embodiment of the present invention; wherein, Figure 2 A. Longitudinal particle size statistical distribution Figure 2 B. Statistical distribution of transverse particle size.

[0021] Figure 3The images show the UV-Vis absorption spectra and circular dichroism (CD) spectra of R-configuration and S-configuration chiral threaded gold nanorods in this invention embodiment; wherein, Figure 3 A represents the UV-vis absorption spectrum. Figure 3 B represents a circular dichroism.

[0022] Figure 4 These are scanning electron microscope (SEM) images of R-BAZN at different 2-aminobenzimidazole dosage ratios in embodiments of the present invention; wherein, Figure 4 A represents a usage ratio of 3%. Figure 4 B represents a usage ratio of 10%. Figure 4 C represents the dosage ratio of 30%.

[0023] Figure 5 The UV-Vis absorption spectra of R-BAZN under different 2-aminobenzimidazole dosage ratios in the embodiments of the present invention are shown.

[0024] Figure 6 This invention presents the characterization and SERS performance comparison of R-BAZN with different ZIF-8-NH2 shell thicknesses in this embodiment; wherein, Figure 6 A~ Figure 6 SEM images of R-BAZN with different shell thicknesses. Figure 6 E~ Figure 6 H-shell thickness statistical distribution map Figure 6 I and Figure 6 SERS spectra of R-BAZN with different shell thicknesses. Figure 6 K and Figure 6 Comparison of SERS spectra of R-BAZN recognizing D / L-lysine enantiomers with different shell thicknesses.

[0025] Figure 7 These are transmission electron microscope (TEM) images and energy dispersive spectral distribution (EDS-Mapping) diagrams of R-BAZN in embodiments of the present invention; wherein, Figure 7 A is a TEM image. Figure 7 B. High-angle annular dark-field scanning transmission image (HAADF-STEM). Figure 7 C represents the surface distribution of Au element. Figure 7 D represents the surface distribution of Zn elements. Figure 7 E represents the surface distribution of element N.

[0026] Figure 8 The images show the Fourier transform infrared (FT-IR) spectra of each intermediate during the R-BAZN synthesis process in this embodiment of the invention.

[0027] Figure 9This is a comparison of X-ray photoelectron spectroscopy (XPS) characterization of R-BAZN and R-AuNRs in the embodiments of the present invention.

[0028] Figure 10 This is a schematic diagram of the structure of the chiral threaded gold nanorods@ZIF-8-NH2 gold film SERS substrate with adjustable gap according to an embodiment of the present invention.

[0029] Figure 11 The images show the SERS spectra of each intermediate during the R-BAZN synthesis process in this embodiment of the invention.

[0030] Figure 12 The SERS spectra and signal uniformity analysis results of 60 random test points on the surface of the NPoM / R-BAZN substrate in this embodiment of the invention are shown.

[0031] Figure 13 The present invention provides SERS spectra and quantitative analysis results of the NPoM / R-BAZN substrate recognizing lysine enantiomers in embodiments of the present invention; wherein, Figure 13 SERS spectra of lysine enantiomers with different ee values. Figure 13 The standard curve of linear fitting between the relative intensity of the B characteristic peak and the ee value.

[0032] Figure 14 The SERS spectra of the NPoM / R-BAZN substrate recognizing six chiral amino acid enantiomers (D / L-isoleucine, D / L-asparagine, D / L-serine, D / L-proline, D / L-leucine and D / L-alanine) in this embodiment of the invention are shown. Detailed Implementation

[0033] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0034] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0035] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0036] This invention addresses existing problems by providing a chiral threaded gold nanorod@ZIF-8-NH2 gold film SERS substrate with adjustable gap, its preparation method, and its application.

[0037] Example 1 This embodiment provides a method for preparing a chiral threaded gold nanorod@ZIF-8-NH2 gold film SERS substrate with adjustable gap, including the following steps: (1) Take a single-crystal silicon wafer with dimensions of 0.3 cm × 0.3 cm, and ultrasonically clean it for 15 minutes each in acetone, anhydrous ethanol, and ultrapure water, then dry the surface with high-purity nitrogen. Place the clean single-crystal silicon wafer as a substrate on the sample stage of the ion sputtering instrument. Evacuate the sputtering chamber to a vacuum level of 5.0 × 10⁻⁶. 4 A gold target with a purity of 99.999% was used as the sputtering source. The sputtering operating voltage was set to 100 V, the sputtering current to 25 mA, and the deposition time to 200 seconds, resulting in an AuF film with a thickness of approximately 100 nm on the surface of a single-crystal silicon wafer.

[0038] (2) Gold nanorods were synthesized using a seed-mediated growth method. The specific steps are as follows: (2.1) Preparation of seed solution: Under stirring at 500 rpm, 5 mL of 0.5 mM HAuCl4 solution and 5 mL of 0.2 M hexadecyltrimethylammonium bromide (CTAB) solution were mixed in a 20 mL glass bottle to obtain a mixed solution. 0.6 mL of 10 mM NaBH4 ice-water solution (0℃) was prepared and added to the above mixed solution under vigorous stirring at 1200 rpm. The solution color changed from yellow to brown. Stirring was continued for 2 min and then stopped. The resulting seed solution was aged in a 30℃ water bath for 2 h for later use.

[0039] (2.2) Preparation of growth solution and synthesis of gold nanorods: Weigh 1.4 g CTAB and 0.25 g sodium oleate (NaOL) into a 150 mL round-bottom flask, add 50 mL ultrapure water, and stir in a 50 °C water bath until completely dissolved. After cooling to 30 °C, add 3.6 mL of 4 mM AgNO3 solution, gently shake for 30 s to mix, and let stand in a 30 °C water bath for 15 min. Then add 50 mL of 1 mM HAuCl4 solution. After stirring for 90 min (700 rpm), the solution becomes colorless. Add 0.6 mL of HCl (37 wt%, 12.1 M) to adjust the pH, and continue stirring at 400 rpm for 15 min. Add 1.25 mL of 0.064 M ascorbic acid (AA) and stir vigorously (700 rpm) for 30 s to obtain the growth solution. Finally, 0.08 mL of the seed solution prepared in step (2.1) was injected into the growth solution, stirred for 30 s, and then allowed to stand at 30 °C for 12 h. The product was collected by centrifugation at 7000 rpm for 10 min.

[0040] (2.3) The preparation process of chiral threaded gold nanorods (RB-Au NRs) is as follows: (2.3.1) Preparation of chiral inducer / hexadecyltrimethylammonium chloride (CTAC) solution: In a 150 mL Erlenmeyer flask, weigh 35.55 mg of R-(+)-1,1'-bi-2-naphthylamine (R-BINAM) and 1.6 g of CTAC, add 50 mL of water, heat at 60 °C with stirring until completely dissolved, and cool to room temperature for later use. The concentrations of CTAC and R-BINAM are 100 mM and 2.5 mM, respectively.

[0041] (2.3.2) Washing of nanoparticles: 10 mL of gold nanorod solution was first washed twice with 10 mL of 10 mM CTAC, then washed twice with R-BINAM / CTAC solution, and finally the particles were redispersed in R-BINAM / CTAC solution; after aging overnight before use, nanoparticle colloids were obtained.

[0042] (2.3.3) Synthesis of chiral spiral gold nanorods: In a 50 mL round-bottom flask, 300 µL of 50 mM tetrachloroauric acid solution was added to a mixture of 21 mL ultrapure water and 6 mL R-BINAM / CTAC solution. The mixture was stirred at 500 rpm for 30 s and then allowed to stand at room temperature for 5 min. Subsequently, 300 µL of the nanoparticle colloid obtained in step (2.3.2) was added, and 600 µL of 0.8 M ascorbic acid was rapidly injected. After vigorous shaking, the mixture was allowed to stand for 20 min. After the reaction was completed, the mixture was centrifuged at 3000 rpm for 10 min, and this process was repeated twice. The precipitate was redispersed in 10 mL of 10 mM CTAC solution to obtain R-configuration chiral spiral gold nanorods (RB-Au NRs), which were stored at 4 °C.

[0043] (3) The preparation process of RB-Au NRs@ZIF-8-NH2 (R-BAZN) is as follows: The prepared RB-Au NRs were washed thoroughly by centrifugation (3000 rpm, 10 min) and immediately redispersed with ultrapure water (prepared fresh for immediate use to avoid aggregation due to prolonged storage) to obtain an RB-Au NRs dispersion. 10 mL of the RB-Au NRs dispersion and 360 µL of 1 mM CTAB solution were added sequentially to a reaction vessel and mixed thoroughly. Then, 10 mL of a mixed ligand solution was added, containing 0.924 M 2-methylimidazole and 3% (by mass) 2-aminobenzimidazole. The mixture was stirred at 400 rpm for 5 min. Next, 10 mL of a 16.8 mM Zn(NO3)2 aqueous solution was added, and stirring was continued for 5 min to promote coordination between the metal ions and the organic ligands. After standing for 90 min, the R-BAZN complex was obtained. Methanol was then added, and the mixture was centrifuged at 3500 rpm for 5 min. The washing was repeated twice, and the mixture was reconstituted in methanol and stored at 4°C for later use.

[0044] (4) Assembly of NPoM / R-BAZN substrate, the specific steps are as follows: The R-BAZN composite was dispersed in methanol to obtain a composite suspension. 6 μL of the suspension was pipetted vertically onto the surface of the AuF film prepared in step (1). The suspension was allowed to stand at 25°C for 10 minutes to allow the nanoparticles to self-assemble on the AuF surface under surface tension. The film was then dried in a 30°C oven for 10 minutes to obtain chiral threaded gold nanorods with adjustable spacing @ZIF-8-NH2 / gold film SERS substrate (NPoM / R-BAZN), as shown in the schematic diagram below. Figure 10 As shown.

[0045] Example 2 The difference between this embodiment and Example 1 is that the amount of 2-aminobenzimidazole is replaced with 10% of the mass of 2-methylimidazole.

[0046] Example 3 The difference between this embodiment and Example 1 is that the amount of 2-aminobenzimidazole is replaced with 30% of the mass of 2-methylimidazole.

[0047] Example 4 The difference between this embodiment and embodiment 1 is that the amount of CTAB solution used in step (3) is 180µL.

[0048] Example 5 The difference between this embodiment and embodiment 1 is that the amount of CTAB solution used in step (3) is 720µL.

[0049] Example 6 The difference between this embodiment and embodiment 1 is that the amount of CTAB solution used in step (3) is 0µL.

[0050] Example 7 The difference between this embodiment and embodiment 1 is that R-BINAM in step (2.3.1) is replaced with S-BINAM.

[0051] Comparative Example 1 The difference between this comparative example and Example 1 is that 2-aminobenzimidazole was not added.

[0052] The above embodiments detail the complete preparation process of the chiral threaded gold nanorods with adjustable gaps@ZIF-8-NH2 / gold film SERS substrate (NPoM / R-BAZN); the intermediates and the final structure were systematically characterized as follows, and the characterization results are as follows. Figures 1 to 11 As shown.

[0053] The morphology of gold nanorods and chiral spiral gold nanorods was characterized by scanning electron microscopy (SEM). The results showed that the gold nanorods had a rod-like structure, and after chiral induction modification, the surface exhibited a clear spiral chiral structure. The R and S configurations were mirror-symmetric. Specific SEM images are shown below. Figure 1 As shown in the figure. The transverse and longitudinal particle sizes of 100 nanoparticles were statistically analyzed, resulting in the particle size distribution statistics shown in the figure. Figure 2 As shown, the gold nanorods have a longitudinal particle size of approximately 145 nm, a transverse particle size of approximately 77 nm, and an aspect ratio of approximately 1.9. The ultraviolet-visible absorption spectrum (UV-Vis) is also shown. Figure 3 A) shows that the chiral spiral gold nanorods exhibit characteristic longitudinal and transverse plasmon resonance absorption peaks; circular dichroism spectroscopy ( Figure 3 B) The R-configuration and S-configuration chiral threaded gold nanorods exhibit mirror-symmetric CD signals near 560 nm, confirming the successful construction of their intrinsic chiral optical activity.

[0054] Figure 4 Images A, 4B, and 4C are scanning electron microscope (SEM) images of R-BAZN obtained with 3%, 10%, and 30% 2-aminobenzimidazole content, respectively. The results show that at 3% and 10% content, R-BAZN maintained a complete core-shell structure and good dispersibility, with smooth nanoparticle surfaces and uniform shell coating. However, when the content increased to 30%, the MOF (ZIF-8-NH2) shell structure significantly collapsed. This is due to the high steric hindrance effect of the benzene ring in 2-aminobenzimidazole and its competitive coordination with 2-methylimidazole; excessive introduction leads to structural distortion of the ZIF-8 framework and a decrease in crystallinity.

[0055] Figure 5The UV-Vis absorption spectra of R-BAZN are shown for different proportions of 2-aminobenzimidazole. The results indicate that as the proportion of 2-aminobenzimidazole increases from 3% to 30%, the maximum absorption wavelength of the complex gradually red-shifts. This is attributed to the fact that the refractive index of the ZIF-8-NH2 shell is greater than that of water. The increase in shell thickness and the introduction of 2-aminobenzimidazole alter the effective refractive index of the medium surrounding the nanoparticles, thereby causing a red-shift of the plasmon resonance peak.

[0056] Taking into account both the requirements for shell structure integrity and chiral recognition performance, a dosage ratio of 10% of 2-aminobenzimidazole was selected as the optimal condition. At this point, sufficient NH2 active groups are introduced to enhance hydrophilicity and interaction with amino acid enantiomers, while ensuring the integrity and uniformity of the ZIF-8-NH2 shell structure.

[0057] In the above embodiments, the ZIF-8-NH2 shell serves as a nanospacer layer, constructing a uniform nanogap (5~16 nm) with controllable size between the chiral threaded gold nanorods and AuF. This gap, acting as a plasma hotspot, can generate extremely strong local electromagnetic field enhancement.

[0058] Figure 6 E to Figure 6 H represents the statistical distribution of shell thickness. With a fixed 2-aminobenzimidazole dosage of 10% of the 2-methylimidazole mass, the thickness of the ZIF-8-NH2 shell could be precisely controlled by adjusting the amount of CTAB added to 0 μL, 180 μL, 360 μL, and 720 μL, respectively. Statistical results showed that the corresponding shell thicknesses were 15.9 ± 2.9 nm, 11.8 ± 1.9 nm, 8.1 ± 1.2 nm, and 5.1 ± 1.0 nm, respectively, exhibiting a uniform distribution. Since the ZIF-8-NH2 shell directly acts as a nanospacer between the chiral threaded gold nanorods and the AuF film layer in the NPoM structure, the aforementioned shell thickness corresponds to the width of the nanospace, indicating that adjusting the amount of CTAB can effectively control the nanospace size, thereby achieving the tunable construction of plasmon "hot spots" in the NPoM structure. Figure 6 I shows the SERS spectra of R-BAZN with different shell thicknesses. The results show that the SERS signal enhancement effect is best when the thickness is 5.1 ± 1.0 nm. Figure 6 J). Figure 6 K represents a comparison of SERS spectra of R-BAZN recognizing D / L-lysine enantiomers with different shell thicknesses. The results show that the enantiomer discrimination is highest when the shell thickness is 8.1±1.2 nm. Figure 6 L).

[0059] Figure 7The images show transmission electron microscopy (TEM) images of R-BAZN and energy dispersive spectral distributions (EDS-Mapping) of Au, Zn, and N elements. The TEM images reveal a ZIF-8-NH2 shell uniformly coating the surface of the chiral threaded gold nanorods, with a clear core-shell structure and a shell thickness of approximately 15 nm. The EDS-Mapping results show that Au is concentrated in the core region, while Zn and N are uniformly distributed throughout the nanoparticle region, confirming the successful coating of the ZIF-8-NH2 shell.

[0060] Figure 8 The images show the Fourier transform infrared (FT-IR) spectra of the intermediates involved in the synthesis of R-BAZN. RB-Au NRs at 2924 cm⁻¹ -1 and 2852 cm -1 The characteristic absorption peak of CTAB is observed at 996 cm⁻¹. Further comparison of the FT-IR spectra of RB-AuNRs@ZIF-8 and RB-AuNRs@ZIF-8-NH₂ reveals that both exhibit a peak at 996 cm⁻¹. -1 The imidazole ring exhibits bending and stretching vibrations at 1146 cm⁻¹. -1 The appearance of a CN stretching vibration peak at 1268 cm⁻¹ confirms the successful encapsulation of the ZIF-8 framework. The key difference between the two is that RB-AuNRs@ZIF-8-NH₂ exhibits a peak at 1268 cm⁻¹. -1 917 cm -1 and 849 cm -1 Three unique characteristic absorption peaks appear at the specified wavenumbers, while RB-AuNRs@ZIF-8 shows no significant absorption at the aforementioned wavenumbers. Specifically, at 1268 cm⁻¹... -1 The absorption peak at 917 cm⁻¹ is attributed to the coupling of the stretching vibration of CN and the in-plane bending vibration of NH in 2-aminobenzimidazole. -1 and 849 cm -1 The absorption peaks at the location are attributed to the out-of-plane deformation vibration of CH in the benzimidazole ring. The appearance of these characteristic peaks indicates that 2-aminobenzimidazole has been successfully incorporated into the ZIF-8-NH2 skeleton.

[0061] Figure 9 Comparison of X-ray photoelectron spectroscopy (XPS) characterizations of R-BAZN and R-AuNRs. The Zn 2p spectrum of R-BAZN shows Zn at 1021.8 eV and 1044.9 eV. 2+ The characteristic peaks of the Zn-N coordination bond were observed in the N 1s spectrum at 398.8 eV, further confirming the successful coating of the ZIF-8-NH2 shell.

[0062] Figure 11The SERS spectra of all intermediates in the R-BAZN synthesis are shown. The SERS spectrum of gold nanorods (Au NRs) shows the characteristic vibrational peaks of CTAB molecules. Chiral threaded gold nanorods (RB-Au NRs) exhibit BINAM-induced fingerprint characteristic peaks, with some peak positions shifted, indicating successful adsorption of chiral molecules. After coating with a ZIF-8-NH2 shell (R-BAZN), the SERS spectrum at 1002 cm⁻¹... -1 and 622 cm -1 Two new characteristic peaks appeared at the specified wavenumber, while RB-Au NRs and RB-Au NRs@ZIF-8 showed no significant SERS response at the aforementioned wavenumber. Specifically, at 1002 cm⁻¹... -1 The absorption peak at 622 cm⁻¹ is attributed to the coupling of the in-plane bending vibration of CH and the stretching vibration of CN in the benzimidazole ring of 2-aminobenzimidazole. -1 The absorption peaks at these locations are attributed to the skeletal deformation vibrations of the benzimidazole ring. The appearance of these two characteristic peaks indicates that 2-aminobenzimidazole has been successfully incorporated into the ZIF-8-NH2 skeleton.

[0063] Performance testing To evaluate the SERS signal uniformity of the NPoM / R-BAZN substrate, the following steps are specifically included: The SERS characteristic peaks of the substrate itself were used directly as Raman signals, without the need for external probe molecules. Sixty test points were randomly selected at different locations on the substrate surface. SERS spectra were acquired under conditions of 785 nm laser excitation, 10-second integration time, and one accumulation. Characteristic peaks (e.g., 1002 cm⁻¹) belonging to the ZIF-8-NH₂ shell were used as the Raman signal. -1 Or 622 cm -1 The intensity of the characteristic vibrations (attributed to 2-aminobenzimidazole) was used as an index to evaluate signal uniformity. The results are as follows: Figure 12 As shown, the 60 random SERS spectra exhibit highly consistent characteristic peak intensities and positions, with the relative standard deviation (RSD) of the selected characteristic peak intensities being less than 6%, confirming the excellent signal uniformity and reproducibility of the NPoM / R-BAZN substrate. These results demonstrate that the NPoM / R-BAZN substrate constructed in this invention features uniform assembly and controllable gaps, possessing significant potential for practical applications.

[0064] application This embodiment details the application of the NPoM / R-BAZN substrate in the recognition of chiral amino acid enantiomers.

[0065] The SERS detection process for chiral amino acid enantiomers on the NPoM / R-BAZN substrate includes the following steps: Using lysine enantiomers as model molecules, the quantitative detection performance of this SERS substrate for chiral amino acids was systematically investigated, including enantiomer recognition ability and quantitative analysis ability of enantiomer excess (ee) value. At the same time, six chiral amino acids, namely D / L-isoleucine, D / L-asparagine, D / L-serine, D / L-proline, D / L-leucine and D / L-alanine, were used as validation objects to examine the universality of this substrate in the recognition of multiple chiral amino acids.

[0066] (1) Identification of lysine enantiomers and quantitative analysis of ee values The chiral recognition system was prepared as follows: 50 μL of R-BAZN complex suspension, 50 μL of 0.1 mmol / L lysine enantiomer aqueous solution (pH=9), and 100 μL of ultrapure water were mixed and allowed to stand at room temperature for 5 min to obtain a mixed solution. Then, 6 μL of the mixed solution was vertically added to the surface of an AuF thin film and dried in a 30℃ oven to obtain the NPoM / R-BAZN detection platform. SERS spectra were acquired using a 785 nm laser (30 mW power) with an integration time of 10 seconds. The differences in SERS spectra between L-lysine and D-lysine at the characteristic peaks were compared.

[0067] To verify the quantitative analytical capability, a series of lysine enantiomer mixtures with different ee values ​​(-100% to 100%) were prepared, with a fixed total concentration of 0.1 mmol / L. The SERS spectra were processed and measured according to the method described above, and calibration curves were plotted using the characteristic peak intensity as an indicator. Figure 13 A is a superimposed SERS spectrum of lysine enantiomers with different ee values. As the ee value changes from -100% to 100%, the intensity of the characteristic peaks shows a regular change. Figure 13 B is the linear fitting curve of the relative intensity of the characteristic peak and the ee value. The results show a good linear relationship in the range of -100% to 100% (Ri). 2 =0.995), indicating that this substrate can be used for accurate quantitative analysis of lysine enantiomer ee values.

[0068] (2) Validation of the universality of the substrate: Identification of the other 6 chiral amino acids To verify the universality of the NPoM / R-BAZN substrate in recognizing chiral amino acids, SERS detection was performed on enantiomers of isoleucine (Ile), asparagine (Asn), serine (Ser), proline (Pro), leucine (Leu), and alanine (Ala) under the same conditions. The specific procedure was as follows: 50 μL of R-BAZN complex suspension, 50 μL of 0.1 mmol / L L-configuration or D-configuration amino acid aqueous solution (pH 9.0), and 100 μL of ultrapure water were mixed and allowed to stand at room temperature for 5 min to obtain a mixed solution. 6 μL of the mixed solution was dropped onto the surface of an AuF thin film, dried in an oven at 30 °C, and then SERS spectra were collected. All spectra were acquired under conditions of 785 nm laser excitation, 30 mW power, and a 10-second integration time. Figure 14 SERS spectra of the six chiral amino acid enantiomers identified by the NPoM / R-BAZN substrate were obtained. The results showed that for each tested amino acid, the L-configuration and D-configuration exhibited significant spectral differences, with distinguishable differences in characteristic peak positions and relative intensities, indicating that the substrate possesses excellent enantiomer recognition ability for multiple chiral amino acids. These results fully demonstrate the universality of the NPoM / R-BAZN substrate in the recognition of multiple chiral amino acid enantiomers.

[0069] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A chiral threaded gold nanorod@ZIF-8-NH2 gold film SERS substrate with adjustable gap, characterized in that, From bottom to top, it includes a substrate, an AuF film, and a chiral threaded gold nanorod@ZIF-8-NH2 composite structure layer; The chiral threaded gold nanorod@ZIF-8-NH2 composite structure layer includes a chiral threaded gold nanorod core and a ZIF-8-NH2 shell coating on its surface; The ZIF-8-NH2 shell forms a nano-gap between the chiral threaded gold nanorod and the AuF film. The size of the nano-gap can be adjusted by controlling the thickness of the ZIF-8-NH2 shell.

2. The chiral threaded gold nanorods@ZIF-8-NH2 gold film SERS substrate with adjustable gap according to claim 1, characterized in that, The thickness of the AuF film is 50~200 nm; the substrate is monocrystalline silicon.

3. The chiral threaded gold nanorods with adjustable gaps@ZIF-8-NH2 gold film SERS substrate according to claim 1, characterized in that, The thickness of the ZIF-8-NH2 shell is 3~30 nm.

4. The chiral threaded gold nanorods@ZIF-8-NH2 gold film SERS substrate with adjustable gap according to claim 1, characterized in that, The surface of the chiral threaded gold nanorod has a threaded chiral structure; wherein the chiral structure is an R configuration or an S configuration; the length of the chiral threaded gold nanorod is 120~200 nm and the diameter is 40~100 nm.

5. The method for preparing the chiral threaded gold nanorods@ZIF-8-NH2 gold film SERS substrate with adjustable gap as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1: AuF film is deposited by plasma sputtering on a pretreated, clean substrate. S2: Chiral spiral gold nanorods were obtained by surface modification with chiral inducing agents. S3: The chiral threaded gold nanorods are dispersed in a Zn(NO3)2 reaction system containing 2-methylimidazole and 2-aminobenzimidazole, and ZIF-8-NH2 is coated on the surface of the chiral threaded gold nanorods by coordination self-assembly to obtain the chiral threaded gold nanorod@ZIF-8-NH2 complex. S4: Assemble the chiral threaded gold nanorods@ZIF-8-NH2 composite onto the surface of the AuF film, and dry it to obtain the chiral threaded gold nanorods@ZIF-8-NH2 gold film SERS substrate with adjustable gap.

6. The preparation method according to claim 5, characterized in that, In step S1, the pretreatment includes: sequential ultrasonic cleaning with acetone, anhydrous ethanol, and ultrapure water, followed by drying; sputtering deposition process: vacuum level below 1×10⁻⁶. -3 Pa, sputtering voltage of 90~110 V, sputtering current of 10~40 mA, and deposition time of 100~500 seconds.

7. The preparation method according to claim 5, characterized in that, In step S2, the preparation process of the chiral threaded gold nanorods specifically includes: S2.1: While stirring, mix chloroauric acid solution with hexadecyltrimethylammonium bromide solution, add sodium borohydride solution prepared with ice water, stir and react, then age to obtain seed solution; S2.2 Dissolve hexadecyltrimethylammonium bromide and sodium oleate in ultrapure water, add silver nitrate solution, chloroauric acid solution, hydrochloric acid and ascorbic acid in sequence and mix well, add the seed solution, let the reaction stand, collect by centrifugation, and obtain gold nanorods; S2.

3. Prepare a chiral induction mixed solution by dissolving the chiral inducing agent and hexadecyltrimethylammonium chloride in water; wash and disperse the gold nanorods with the chiral induction mixed solution, then add chloroauric acid solution and ascorbic acid to react, collect by centrifugation, and obtain chiral spiral gold nanorods; The chiral inducer is R-(+)-1,1'-bi-2-naphthylamine or S-(-)-1,1'-bi-2-naphthylamine.

8. The preparation method according to claim 5, characterized in that, Step S3 specifically includes: centrifuging and washing the chiral threaded gold nanorods and redispersing them in ultrapure water, then sequentially adding hexadecyltrimethylammonium bromide solution, 2-methylimidazolium / 2-aminobenzimidazole mixed solution and Zn(NO3)2 aqueous solution, stirring and reacting, allowing to stand, centrifuging and washing to obtain the chiral threaded gold nanorods@ZIF-8-NH2 composite.

9. The preparation method according to claim 5, characterized in that, In step S4, the chiral threaded gold nanorods@ZIF-8-NH2 composite is dispersed in ultrapure water to obtain a composite suspension; 2~15 μL of the composite suspension is vertically dropped onto the surface of the AuF film and dried at 25~35℃ to obtain the chiral threaded gold nanorods@ZIF-8-NH2 gold film SERS substrate with adjustable gap.

10. The application of the chiral threaded gold nanorods@ZIF-8-NH2 gold film SERS substrate with adjustable gap as described in any one of claims 1 to 4, or the chiral threaded gold nanorods@ZIF-8-NH2 gold film SERS substrate with adjustable gap prepared by the preparation method described in any one of claims 5 to 9, in the field of non-aromatic amino acid molecular detection, characterized in that, The non-aromatic amino acid molecule is at least one of lysine, isoleucine, asparagine, serine, proline, leucine, and alanine.

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