A SERS substrate based on mesoporous Au nanospheres and its preparation method and application

Abstract

The present invention belongs to the technical field of laser Raman spectroscopy detection and relates to a SERS substrate based on mesoporous Au nanospheres, a preparation method and application thereof. Nα-[(9H-fluorene-9-ylmethoxy)carbonyl]-D-lysine hydrochloride is used as a template agent, and mesoporous Au nanospheres with three-dimensional radial shapes are synthesized by a one-pot co-reduction method. The prepared SERS substrate based on the mesoporous Au nanospheres can achieve non-contact enhancement and retention of molecular intrinsic vibration information, selectively enrich small molecule enantiomers, eliminate interference from biological matrix macromolecules, and can achieve trace detection of MA and METH in urine.

Description

Technical Field

[0001] The invention belongs to the technical field of laser Raman spectroscopy detection, and relates to a SERS substrate based on mesoporous Au nanospheres, and a preparation method and application thereof. Background Art

[0002] Enantiomers of chiral molecules exhibit significant differences in biological activity, pharmacological effects, and metabolic pathways. For example, mandelic acid (MA), a key chiral synthon, R-MA is a key precursor to the anticholinergic drug glycopyrrolate, while S-MA is used in the synthesis of the antibiotic tropine mandelate. Racemic MA can also be illicitly converted into phenylethylamine drugs. Taking illicit drug abuse detection as another example, dextrorotatory methamphetamine (METH) is a central nervous system stimulant. The levorotatory form is a drug approved for cardiovascular treatment, but has a weaker central nervous system stimulant effect. Furthermore, some clinical drugs, such as selegiline (SLG), a prescription drug for Parkinson's disease, are metabolized to dextrorotatory METH in the body. This has led to controversy in drug use and driving under the influence detection cases, where suspects who test positive for SLG in urine often claim to have taken SLG. Therefore, enantiomeric identification is crucial for drug safety and understanding the metabolic pathways of chiral substances. Their significant pharmacological differences require reliable and rapid chiral recognition methods to provide a scientific basis for distinguishing legal from illicit drugs.

[0003] Currently, chiral recognition technologies primarily encompass traditional circularly polarized light chiral recognition and molecular imprinting, depending on their molecular mechanisms of action. The first approach relies on the inherent optical activity of chiral molecules and their selective absorption or scattering of circularly polarized light. This approach suffers from insufficient sensitivity and is susceptible to interference from complex matrices. The second approach relies on traditional lock-key molecular recognition, employing chiral probes or stationary phases designed to complement the stereochemistry of the target molecule and leveraging intermolecular forces such as hydrogen bonds and hydrophobic interactions to achieve selective recognition. However, this approach suffers from poor stability and limited versatility.

[0004] Surface-enhanced Raman scattering (SERS), a powerful technique with single-molecule sensitivity and high-molecular fingerprint specificity, is widely used in surface analysis and chemical identification in a wide range of fields, including chemistry, biology, physics, and materials science. Studies have shown that SERS substrates based on specially designed noble metal nanostructures can significantly enhance the Raman signal of target molecules through the localized surface plasmon resonance (LSPR) effect, achieving a Raman signal of up to 10 6 -10 14 However, traditional chiral SERS substrates suffer from uneven distribution, resulting in poor signal reproducibility and limited application. Developing new SERS substrates that can enrich small molecule enantiomers and eliminate interference from large molecules in the biological matrix is ​​of great significance for the rapid, accurate, and sensitive identification of illicit drug use. Summary of the Invention

[0005] To overcome the shortcomings of the existing technology, the present invention provides a SERS substrate based on mesoporous Au nanospheres, as well as a preparation method and application thereof. Using Nα-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-lysine hydrochloride (Fmoc-D-Lys-OH·HCl) as a template, Au nanospheres with three-dimensional radial mesopores are synthesized by a one-pot reduction method. The mesoporous channels can selectively enrich small molecule enantiomers and eliminate interference from large molecules in the biological matrix. This method is simple and time-saving, does not require complex sample pretreatment, and can achieve trace detection of MA and METH in urine.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A first aspect of the present invention provides a method for preparing a SERS substrate based on mesoporous Au nanospheres, comprising the following steps:

[0008] (1) Preparation of template-metal precursor complex: Add Fmoc-D-Lys-OH·HCl solution and chloroauric acid solution to deionized water at 2-4°C in an ice bath and stir to form a template-metal precursor complex solution;

[0009] (2) Synthesis of mesoporous Au nanospheres: Add ascorbic acid solution to the template-metal precursor complex solution in an ice bath at 2-4°C and stir evenly, perform co-reduction reaction for 20-40 min, centrifuge and wash, and redisperse in ultrapure water to obtain a 2-3 mM mesoporous Au nanosphere solution;

[0010] (3) Preparation of SERS substrate: The mesoporous Au nanosphere solution is evenly dropped on the substrate, dried, and then soaked in a mixed solution of acetone and acetic acid with a pH of 3 to 5 for 20 to 24 hours to obtain a SERS substrate.

[0011] Preferably, the concentration of the Fmoc-D-Lys-OH·HCl solution in step (1) is 20-30 mM, more preferably 24.7 mM; the concentration of the chloroauric acid solution is 45-55 mM, more preferably 50 mM.

[0012] Preferably, in step (1), the volume ratio of the Fmoc-D-Lys-OH·HCl solution, the chloroauric acid solution, and the deionized water is 1-3:0.3-0.8:15-25.

[0013] Preferably, the concentration of the ascorbic acid solution in step (2) is 95-105 mM, and the volume ratio of the ascorbic acid solution to the template-metal precursor complex solution is 1:18-25;

[0014] Preferably, the stirring speed is 800-1200 rpm.

[0015] Preferably, the centrifugal speed in step (2) is 4000-6000 rpm, and the time is 8-15 min.

[0016] Preferably, the substrate in step (3) is a Raman single crystal silicon wafer, and 2-3 μL of the mesoporous Au nanosphere solution is evenly added per square centimeter of the substrate.

[0017] Preferably, the content of acetone in the mixed solution in step (3) is 85% to 95%, and the content of acetic acid is 5% to 15%.

[0018] The second aspect of the present invention provides a SERS substrate prepared by the method described, comprising a substrate and mesoporous Au nanospheres that modify the substrate, wherein the mesoporous Au nanospheres have a bayberry-like morphology, a diameter of 200±25 nm, and a mesoporous structure with three-dimensional radial open pores on the surface, with a pore diameter of 1.5-2.0 nm.

[0019] The third aspect of the present invention provides the use of the SERS substrate in the identification and detection of mandelic acid.

[0020] A fourth aspect of the present invention provides the use of the SERS substrate in the detection of trace amounts of methamphetamine.

[0021] The present invention's method for preparing a SERS substrate based on mesoporous Au nanospheres successfully constructs a mesoporous structure with three-dimensional radially open pores through a template-free, seed-free, one-pot reduction system controlled by the dynamic self-assembly of surfactant molecules. This innovative design exhibits three synergistic advantages: first, a precisely controllable and uniform structure. The size and distribution density of the mesoporous channels can be precisely adjusted by the ascorbic acid solution concentration and reaction kinetics, forming high-density, uniformly ordered "hotspots"; second, a selective molecular sieving function. The open pore network, through the pore confinement effect, allows only specific target molecules to efficiently penetrate the particle interior, significantly improving the substrate's anti-interference ability in complex biological matrices; third, an in situ chiral functionalization strategy. The chiral ligand Fmoc-D-Lys-OH·HCl solution is introduced during the mesopore growth process. The π-π stacking and hydrogen bonding synergistic effect achieves the directed self-assembly of chiral recognition sites, ensuring the stability of the chiral surface interface conformation and avoiding the loss of active sites caused by traditional post-modification processes. This SERS substrate can realize the detection of MA and trace amounts of METH in urine without interference from large molecules in the biological matrix.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The present invention adopts a one-pot co-reduction method to synthesize mesoporous Au nanosphere solution. The synthesis method is simple and effective, and the pore structure is controllable. The mesopore size is 1.5-2 nm.

[0024] (2) The method of the present invention prepares a SERS substrate based on mesoporous Au nanospheres, which can achieve non-contact enhancement, avoid direct chemical bonding, and retain the inherent vibration information of molecules; the mesoporous channels selectively enrich small molecule enantiomers, eliminate interference from large molecules in the biological matrix, and have a significant nano-confinement effect.

[0025] (3) The SERS substrate based on mesoporous Au nanospheres of the present invention has a detection limit of 0.1 mM for MA and an enantioselectivity of 25 ee%. It also has good anti-interference ability for the detection of METH in urine, and the minimum detection concentration of METH is 5 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 These are the characterization and analysis results of the template-metal precursor complexes prepared in Example 1 and Comparative Example 1, where a is Example 1 and b is Comparative Example 1.

[0027] Figure 2 This is the XPS graph of the template-metal precursor complex prepared in Example 1.

[0028] Figure 3 1s spectra of the Fmoc-D-Lys-OH·HCl and the template-metal precursor complex in Example 1, a is the N 1s spectrum of Fmoc-D-Lys-OH·HCl, and b is the N 1s spectrum of the template-metal precursor complex.

[0029] Figure 4 These are characterization analysis diagrams of the mesoporous Au nanospheres prepared in Example 1, a and b are low-magnification TEM images of the mesoporous Au nanospheres, and c and d are high-resolution HAADF-STEM images of the mesoporous Au nanospheres.

[0030] Figure 5 These are the CD analysis results of the mesoporous Au nanospheres prepared in Example 1 and Comparative Example 1. Figure a is the CD spectrum of Fmoc-D-Lys-OH·HCl and Fmoc-Lys-OH·HCl solutions, and Figure b is the CD spectrum of the mesoporous Au nanosphere solution synthesized by inducing Fmoc-D-Lys-OH·HCl and Fmoc-Lys-OH·HCl solutions.

[0031] Figure 6 This is a stability analysis diagram of the SERS substrate based on mesoporous Au nanospheres prepared in Example 1 in 2,6-DMPI.

[0032] Figure 7These are the SERS enhancement and MA enantiomer identification analysis diagrams of the SERS substrate based on mesoporous Au nanospheres prepared in Example 1 after being soaked in acetone and acetic acid solutions for different time periods (Au / D1 not soaked, Au / D2 soaked for 24 hours, Au / D3 soaked for one week); wherein a is the SERS spectrum analysis diagram of the SERS substrates of Au / D1, Au / D2, and Au / D3; b is the SERS enantiomer identification analysis diagram of the SERS substrates after cleaning for different time periods.

[0033] Figure 8 Figures a, b, c, and d are the selective effect analysis diagrams of the SERS substrate based on mesoporous Au nanospheres prepared in Example 1 and Comparative Example 1 on different model molecules. Figures a, b, c, and d are 1.2 nm hemin, 4.4 nm myoglobin, 5 nm hemoglobin, and 2.2 nm FITC-labeled recombinant protein (FITC-Labeled VEGF 165 Protein) and the substrate's selective SERS spectra on them, respectively.

[0034] Figure 9 This is the analysis diagram of the SERS substrate based on mesoporous Au nanospheres prepared in Example 1 for the identification of MA enantiomers, where a is the Raman spectra of R-MA and S-MA alone, and b is the SERS substrate based on mesoporous Au nanospheres for the identification of MA enantiomers.

[0035] Figure 10 These are MA detection limit analysis diagrams of the SERS substrate based on mesoporous Au nanospheres prepared in Example 1, wherein Figure a is the detection limit analysis diagram of the substrate for R-MA, and Figure b is the detection limit analysis diagram of the substrate for S-MA.

[0036] Figure 11 The selective analysis diagram of the SERS substrate based on mesoporous Au nanospheres prepared in Example 1 for MA enantiomers, Figure a is the SERS spectrum of the substrate for different enantiomers (ee%) chiral recognition system, Figure b is the SERS spectrum of I 1002 / I 1022 The relationship between the difference in relative intensity ratio and ee value (%).

[0037] Figure 12 This is an anti-interference analysis diagram of the SERS substrate based on mesoporous Au nanospheres prepared in Example 1 in urine.

[0038] Figure 13 This is a diagram of the SERS substrate based on mesoporous Au nanospheres prepared in Example 1 used to detect and analyze urine samples from real drug users. Figure a shows the SERS results of the substrate detecting urine samples, and Figure b shows the comparison between the LC-MS detection results and the Raman results.

[0039] Figure 14 TEM images of mesoporous Au nanospheres prepared in Example 1 and Comparative Example 2, a corresponds to Example 1, and b corresponds to Comparative Example 2.

[0040] Figure 15 Chirality analysis diagram of the Au NPs and the SERS substrate modified with Fmoc-D-Lys-OH·HCl in Comparative Example 2; Figure a is the MA analysis result of the SERS substrate, and Figure b is the PCA analysis result. DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to specific examples and accompanying drawings. In the specific embodiments of the present invention, the methods described are conventional methods in the art unless otherwise specified.

[0042] Example 1

[0043] A method for preparing a SERS substrate based on mesoporous Au nanospheres, comprising the following steps:

[0044] (1) Preparation of template-metal precursor complex: Weigh 25 mg of Fmoc-D-Lys-OH·HCl (CAS No.: 139262-23-0, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and dissolve it in 2.5 mL of ultrapure water. Ultrasonic dissolution is required to obtain 24.7 mM Fmoc-D-Lys-OH·HCl; weigh 1 g of HAuCl4 (CAS No.: 16903-35-8, purchased from Sinopharm Chemical Reagent Co., Ltd.) and dissolve it in 50 mL of ultrapure water to obtain 50 mM HAuCl4 solution. Then, in a conical flask containing 17.5 mL of ultrapure water, add 2.5 mL of Fmoc-D-Lys-OH·HCl solution and 0.5 mL of HAuCl4 solution respectively, and stir evenly to form a template-metal precursor complex solution; the entire reaction process must be carried out in an ice bath (4°C) and a stirring speed of 1000 rpm.

[0045] (2) Synthesis of mesoporous Au nanospheres: Weigh 0.176 g of ascorbic acid and dissolve it in 10 mL of deionized water. This step is vortexed to dissolve to obtain a 0.1 M ascorbic acid solution. Take 1 mL of ascorbic acid solution and quickly add it to the template-metal precursor complex solution. The solution quickly changes from bright yellow to blue-green. Continue the reaction for 30 min, and the color of the solution gradually deepens to obtain a mesoporous Au nanosphere solution. The mesoporous Au nanosphere solution is centrifuged and washed with ultrapure water. Repeat 1-2 times, with a centrifugation time of 10 min and a speed of 5000 rpm. The obtained mesoporous Au nanospheres are then dissolved in 20 mL of ultrapure water to obtain a 2.5 mM mesoporous Au nanosphere solution. This step must also be performed in an ice bath (4°C) and a stirring speed of 1000 rpm.

[0046] (3) Preparation of SERS substrate: 2.5 μL of mesoporous Au nanosphere solution was evenly dropped on a Raman single crystal silicon wafer with an area of ​​1 cm × 1 cm. After drying with an infrared lamp, it was immersed in a mixed solution of acetone and acetic acid (pH = 4, the mass fraction of acetone in the mixed solution of acetone and acetic acid is 85% and the mass fraction of acetic acid is 15%) for 24 h to obtain a SERS substrate.

[0047] Cryo-EM technique was used to characterize the template-metal precursor complex, such as Figure 1 As shown, it can be seen that at this synthesis stage, the self-assembly process of the nanospheres has begun, forming mesoporous nanospheres with certain size and structural characteristics, and the molecules are orderly aggregated and self-assembled. The synthesis process relies on the directional arrangement of micelles to control the pore orientation and the in situ phase transition to achieve mesoporous inheritance.

[0048] In-depth analysis of the chemical state evolution of the template-metal precursor complex was carried out, and XPS tests were performed. Figure 2 As shown, there are two characteristic peaks at 84.0 and 87.6 eV, which are respectively related to the Au 4f 7 / 2 and Au 4f 5 / 2 The Au 4f peaks were further convolved to separate the signals from Au(I) and Au(0). Figure 3 This is the N1s spectrum of the complex of Fmoc-D-Lys-OH·HCl and template-metal precursor. The amide bond of the Fmoc group does not directly participate in the coordination, and its binding energy remains stable. The peak at 402 eV is mainly attributed to the ε-amino group (side chain -NH2) of lysine and the Au in HAuCl4. 3+ Forming a coordination bond (N→Au bond). This indicates that the synthesis process of mesoporous Au nanospheres is as follows: Fmoc-D-Lys-OH·HCl micelles enrich Au through electrostatic and coordination interactions. 3+ , forming Au(I) intermediates, and ascorbic acid preferentially reduces Au at the micelle-solution interface. 3+ , forming Au 0 The crystal nucleus grows epitaxially along the long axis of the micelle and inherits the radial pore structure of the micelle.

[0049] The morphology and mesoporous structure of mesoporous Au nanospheres were systematically characterized based on low-magnification TEM and high-resolution HAADF-STEM. Figure 4 As shown. Figure 4As shown in (a) and (b) below, under low-magnification TEM observation, the mesoporous Au nanospheres exhibit excellent monodispersity, with uniform spacing between nanometers. High-resolution HAADF-STEM images of the mesoporous Au nanospheres (c) and (d) further reveal the fine structure of the pores. The mesopore openings are relatively uniform and exhibit a stacking pattern, with pore depths of approximately 50-80 nm and clear lattice fringes visible on the inner walls. The pore size distribution primarily ranges from 1.5-2.0 nm. This size of mesopore provides a rich pore structure within the nanospheres, significantly increasing the number of hotspots within the pores and thus greatly enhancing the material's SERS activity.

[0050] The stability of the SERS substrate based on mesoporous Au nanospheres was analyzed. The treated substrate was immersed in a low concentration of 2,6-DMPI ethanol solution, and about 50 SERS spectra were collected. The obtained Raman spectra were visualized by three-dimensional color mapping, as shown in Figure 2. Figure 6 As shown, the enhanced signal is 2.3×10 5 of magnitude.

[0051] Comparative Example 1

[0052] The only difference from Example 1 is that Fmoc-D-Lys-OH·HCl is replaced by Fmoc-L-lysine hydrochloride (Fmoc-Lys-OH·HCl). The characterization analysis diagram of the prepared template-metal precursor complex is as follows: Figure 1 Figure b. As can be seen, the L-configuration is larger overall than the D-configuration. Guided by the D-configuration template, the Fmoc-Lys molecules form a high-density self-assembly through π-π stacking and hydrogen bonding. However, the L-configuration template suffers from steric hindrance, resulting in a lower ligand content on its surface. This is also the reason why the L-configuration template is less effective than the D-configuration template in subsequent MA enantiomer identification.

[0053] The chirality information of the mesoporous Au nanospheres of Example 1 and Comparative Example 1 was further analyzed by CD spectroscopy. Figure 5 As shown in Figure 1, the solutions obtained from Fmoc-D-Lys-OH·HCl and Fmoc-Lys-OH·HCl clearly exhibit opposite chirality in the CD spectra. Figure 1 (b) shows that the mesoporous Au nanospheres synthesized based on the template inducer exhibit similar chirality information as the original solution in the CD spectrum, and there is a trend of signal enhancement. The radial pore structure of the mesoporous Au nanospheres breaks the geometric symmetry, inducing a local electric field to produce a chiral gradient distribution. This special structure promotes the interaction between chiral molecules and the nanosphere surface, thereby enhancing the chiral signal.

[0054] The openness and selectivity of the pores of the mesoporous Au nanospheres prepared in Example 1 (Au-D) and Comparative Example 1 (Au-L) were explored. The specific Raman fingerprint spectrum of the mesoporous Au nanospheres was used to detect four model molecules of different sizes with a common heme functional group, namely 1.2 nm hemin, 2.2 nm FITC-labeled recombinant protein (FITC-Labeled VEGF 165 Protein), 4.4 nm myoglobin, and 5 nm hemoglobin. A certain concentration of solution was prepared, mixed with the material, and then centrifuged and washed thoroughly to remove unbound molecules. The material was then dropped onto a silicon wafer and its Raman spectrum was collected. The experimental results are shown in Figure 2. Figure 8 As shown, only 1.2 nm hemoglobin was completely retained and produced a strong Raman signal, and 2.2 nm FITC-labeled recombinant protein was partially retained, confirming that the mesopores have a size-selective confinement effect.

[0055] Comparative Example 2

[0056] A method for preparing a SERS substrate based on mesoporous Au nanospheres is different from Example 1 only in that step 2) is performed at room temperature of 25°C.

[0057] Figure 14 Figures a and b are TEM images of the mesoporous Au nanospheres prepared in Example 1 and Comparative Example 2, respectively. During the reaction process, it can be seen that the reaction proceeds faster at room temperature and the mesoporous structure of the particles is not obvious. Under ice bath conditions, the particles are smaller and the size distribution is more uniform. This is because low temperature helps control the crystal growth rate and reduce defects.

[0058] Comparative Example 3

[0059] A method for preparing a SERS substrate comprises the following steps:

[0060] 40 nm Au NPs were prepared using the citrate reduction method and modified with Fmoc-D-Lys-OH·HCl on their surface. 100 mL of chloroauric acid solution (0.01 wt% HAuCl4) was added to a round-bottom flask. The above solution was placed on a stirrer and heated to boiling while maintaining reflux. When the solution boiled, 1.0 mL of sodium citrate solution (1 wt%) was quickly added. Sodium citrate, as a reducing agent, can reduce chloroauric acid to Au NPs. The solution was boiled for 30 min to ensure that the reaction was complete. After the reaction was completed, the solution was naturally cooled to room temperature to obtain a well-dispersed Au NPs solution. Subsequently, 1~2 mL of AuNPs solution was mixed with 1~2 mL of 10 mg / mL Fmoc-D-Lys-OH·HCl solution and modified for 30~60 min to obtain an Au nanosphere solution. The SERS substrate was then prepared in the same manner as step (3) of Example 1 to obtain a SERS substrate.

[0061] The chiral analysis of Au NPs and SERS substrate modified with Fmoc-D-Lys-OH·HCl in Example 3 was performed, but the results showed that these substrates did not exhibit the ideal chiral analysis effect. Figure 15 As shown by Figure 15 As can be seen in a, smooth AuNPs can provide a certain SERS enhancement effect, but the MA characteristic peak 1002 cm -1 The position signal was weak, and 50 Raman spectra were subsequently collected for PCA analysis. The spectra showed a high degree of overlap (shown in b). This is due to the lack of chiral features on the surface and the broken symmetry in the structure, which makes it impossible to effectively distinguish between R-MA and S-MA. Even if Fmoc-D-Lys-OH·HCl was modified on the surface of Au NPs, the chiral recognition ability was not significantly improved. This shows that relying solely on surface modification or smooth nanostructures is difficult to achieve efficient chiral differentiation.

[0062] Application Example 1

[0063] The SERS substrate based on mesoporous Au nanospheres of Example 1 was used for MA enantiomer identification and detection, and the steps were as follows:

[0064] 2.5 μL of 10 mM R-MA and 10 mM S-MA aqueous solutions were respectively added dropwise to the SERS substrate and allowed to dry naturally. Raman spectra were collected using a 785 nm laser (power 0.1 mW) (integration time 10 s).

[0065] Study step (3) The effect of immersion time on chiral recognition of SERS substrates was studied. The following experiments were conducted on the SERS substrates: unwashed (Au / D1), washed for 24 hours (Au / D2), and washed for one week (Au / D3). SERS spectra were then analyzed. Figure 7 As shown in Figure 1, a is the SERS spectrum analysis of Au / D1, Au / D2, and Au / D3; b is the SERS substrate discrimination analysis of S-MA and R-MA enantiomers after cleaning for different times. It can be seen that the SERS substrate cleaned for 24 hours exhibits the best SERS discrimination for MA enantiomers, while extended cleaning time leads to signal disorder (carbonization) and loss of chiral function.

[0066] SERS substrate based on mesoporous Au nanospheres for MA enantiomer identification Figure 9 As shown in the figure, a is the Raman spectra of R-MA and S-MA alone, and b is the SERS substrate based on mesoporous Au nanospheres for the identification of MA enantiomers. It can be seen that at 618 cm -1 、826cm -1 、858 cm -1 , 1002 cm -1 , 1174 cm -1 、1197 cm -1 、1537 cm -1 and 1593 cm -1 A new characteristic peak appeared at 1002 cm, which is consistent with the characteristic Raman band of MA. Based on the mesoporous Au nanospheres as SERS substrate, different action modes were produced for R-MA and S-MA enantiomers. -1 and 1593 cm -1 There is a significant difference in the relative intensities of the two spectral bands. When S-MA participates in the chiral recognition system, the 1002 cm -1 and 1593 cm -1 The band intensity of α-MA is much higher than that of R-MA.

[0067] To verify the sensitivity of the SERS substrate based on mesoporous Au nanospheres, R-MA and S-MA solutions with different concentrations of 100 mM, 50 mM, 10 mM, 5 mM, 1 mM and 0.1 mM were prepared, and SERS analysis was performed, e.g. Figure 10 As shown in Figure 1, Figure a shows the sensitivity analysis for R-MA, and Figure b shows the sensitivity analysis for S-MA. As the MA concentration increases, the SERS signal intensity also increases, indicating that the material has good concentration dependence. In particular, even at a low concentration of 0.1 mM, S-MA can still detect a weak but clear MA characteristic signal, indicating that the SERS substrate based on mesoporous Au nanospheres has high detection sensitivity.

[0068] To further explore the enantioselectivity of the SERS substrate based on mesoporous Au nanospheres, MA enantiomer solutions with different enantiomer (25% ee) values ​​were prepared and subjected to SERS analysis, such as Figure 11 As shown, a is the SERS spectrum of the chiral recognition system of different enantiomers (ee%), b is I 1002 / I 1022 The relationship between the difference in relative intensity ratio and ee value (%). It can be seen that with the increase of S-MA concentration in MA enantiomer solution, the main Raman peak at 1002 cm -1 The intensity of the 1002 / I 1022 The relative intensity ratio of the S-MA ( ) as the chiral selectivity internal standard gradually increases with the ee% value of S-MA, indicating that the SERS substrate based on mesoporous Au nanospheres exhibits significant chiral selectivity.

[0069] Application Example 2

[0070] The SERS substrate based on mesoporous Au nanospheres prepared in Example 1 was used for the detection of METH urine traces, and the steps were as follows:

[0071] 1 mL of urine from a drug addict was diluted with 4 mL of ultrapure water and vortexed to mix. 2.5 μL of the diluted urine was added dropwise to the SERS substrate of Example 1 and allowed to stand for 1 min for adsorption. Raman spectra were collected using a 785 nm laser (power 0.1 mW) (integration time 10 s).

[0072] In order to explore the anti-interference ability of the SERS substrate based on mesoporous Au nanospheres on real biological samples, we tested the main components in urine such as urea, uric acid, glucose and other interfering components. Figure 12 As shown in the figure, even after adding 100 mM of interfering components to the substrate, the spectrum did not show the characteristic signals of the interfering components. Affected by the nano-confinement of the material, these molecules did not enter the pores and interact with the substrate.

[0073] The METH concentration in the same urine sample was independently measured using LC-MS technology, and the two results were compared, such as Figure 13As shown in Figure 1, (a) shows the Raman-based METH concentration measurement results, and (b) shows the LC-MS-based METH concentration measurement results. The SERS and LC-MS results show minimal deviation, demonstrating clinical-grade detection reliability in complex biological matrices. This demonstrates high sensitivity and selectivity for METH, a drug of abuse, with a minimum detection concentration of 5 μM. The mesoporous channels of the nanospheres selectively allow METH molecules smaller than the pores to enter while excluding proteins or macromolecules above the cutoff value. This enables direct detection of small molecules in complex biological samples, avoiding tedious sample pretreatment.

[0074] The above description is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several modifications and improvements without departing from the inventive concept, and these all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a SERS substrate based on mesoporous Au nanospheres, characterized in that: The following steps are involved: (1) Preparation of template-metal precursor complex: Mix Nα-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-lysine hydrochloride solution and chloroauric acid solution in an ice bath at 2-4°C, then add deionized water and stir to form a template-metal precursor complex; (2) Synthesis of mesoporous Au nanospheres: Add ascorbic acid solution to the template-metal precursor complex in an ice bath at 2-4°C and stir evenly, perform co-reduction reaction for 20-40 min, centrifuge and wash, and redisperse in ultrapure water to obtain a 2-3 mM mesoporous Au nanosphere solution; (3) Preparation of SERS substrate: The mesoporous Au nanosphere solution is evenly dropped on the substrate, dried, and then soaked in a mixed solution of acetone and acetic acid for 20 to 24 hours to obtain a SERS substrate.

2. The preparation method according to claim 1, characterized in that In step (1), the concentration of the Nα-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-lysine hydrochloride solution is 20-30 mM, and the concentration of the chloroauric acid solution is 45-55 mM.

3. The preparation method according to claim 1, characterized in that In step (1), the volume ratio of the Nα-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-lysine hydrochloride solution, chloroauric acid solution, and deionized water is 1-3:0.3-0.8:15-25.

4. The preparation method according to claim 1, characterized in that The concentration of the ascorbic acid solution in step (2) is 95-105 mM, and the volume ratio of the ascorbic acid solution to the template-metal precursor complex solution is 1:18-25.

5. The preparation method according to claim 1, characterized in that The centrifugal speed in step (2) is 4000-6000 rpm, and the time is 8-15 min.

6. The preparation method according to claim 1, characterized in that The substrate in step (3) is a Raman single crystal silicon wafer, and 2-3 μL of the mesoporous Au nanosphere solution is evenly added per square centimeter of the substrate.

7. The preparation method according to claim 1, characterized in that The pH of the mixed solution in step (3) is 3-5, the mass fraction of acetone in the mixed solution is 85%-95%, and the mass fraction of acetic acid is 5%-15%.

8. The SERS substrate prepared by the method according to any one of claims 1 to 7, characterized in that: The invention comprises a substrate and a mesoporous Au nanosphere modified with the substrate. The mesoporous Au nanosphere has a bayberry-like morphology, a diameter of 200±25 nm, and a mesoporous structure with three-dimensional radial open pores on the surface, and the pore diameter is 1.5-2.0 nm.

9. Use of the SERS substrate as claimed in claim 8 in identification and detection of mandelic acid.

10. Use of the SERS substrate as claimed in claim 8 in trace detection of methamphetamine.

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