SERS (Surface Enhanced Raman Scattering) substrate based on mesoporous Au nanospheres as well as preparation method and application of SERS substrate

Abstract

The invention belongs to the technical field of laser Raman spectrum detection, and relates to an SERS (Surface Enhanced Raman Scattering) substrate based on mesoporous Au nanospheres as well as a preparation method and application of the SERS substrate based on the mesoporous Au nanospheres. N alpha-[(9H-fluorene-9-ylmethoxy) carbonyl]-D-lysine hydrochloride is used as a template agent, and the three-dimensional radial mesoporous Au nanospheres are synthesized through a one-pot co-reduction method; according to the prepared SERS substrate based on the mesoporous Au nanospheres, non-contact enhancement and retention of inherent vibration information of molecules can be realized, micromolecular enantiomers are selectively enriched, interference of biological matrix macromolecules is eliminated, and trace detection of MA and METH in urine can be realized.

Description

Technical Field

[0001] 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 thereof and an application thereof. Background Art

[0002] Enantiomers of chiral molecules show significant differences in biological activity, pharmacological effects and metabolic pathways. For example, mandelic acid (MA) is a key chiral synthon. R-MA is a key precursor of the anticholinergic drug glycopyrronium bromide, while S-MA is used to synthesize the antibiotic tropicamide mandelate, and racemic MA can be illegally converted into phenethylamine drugs. Taking the detection of illegal drug abuse as another example, dextral methamphetamine (METH) is a central nervous system stimulant, and the levorotatory form is a drug approved for the treatment of the cardiovascular system with relatively weak central nervous excitation. Moreover, some clinical drugs such as the prescription drug selegiline (SLG) used for the treatment of Parkinson's disease are metabolized into dextral METH in the body, resulting in disputes in the practice of drug use and drug driving identification, where suspects with positive urine tests often claim to have taken SLG. Therefore, enantiomer discrimination is crucial for drug safety and understanding chiral biological metabolic pathways. Its huge pharmacological differences require reliable and rapid chiral recognition methods to provide a scientific basis for distinguishing legal drug use from illegal drug use.

[0003] Currently, according to different molecular action mechanisms, chiral recognition technologies mainly include traditional circularly polarized light chiral recognition technology and molecular imprinting recognition technology. The first type of method relies on the inherent optical activity of chiral molecules and the selective absorption or scattering characteristics of chiral molecules to circularly polarized light, with insufficient sensitivity and being easily interfered by complex matrices. The second type of method relies on traditional lock-and-key molecular recognition. By designing chiral probes or stationary phases that are complementary to the stereoconfiguration of the target molecule, selective recognition is achieved through intermolecular forces such as hydrogen bonds and hydrophobic interactions, with poor stability and limited generality.

[0004] Surface-enhanced Raman scattering (SERS) technology, as a powerful technology with single-molecule level sensitivity and high molecular fingerprint specificity, has been widely used in surface analysis and chemical identification in a wide range of fields such as chemistry, biology, physics and materials science. Research shows that SERS substrates based on specially designed noble metal nanostructures can significantly enhance the Raman signals of target molecules through the local surface plasmon resonance (LSPR) effect, achieving a Raman signal enhancement of up to 10 6 -10 14 times. However, traditional chiral SERS substrates have problems such as poor signal reproducibility due to uneven distribution and limited application scope. Developing new SERS substrates that can enrich small molecule enantiomers and exclude interference from biological matrix macromolecules is of great significance for the rapid, accurate and sensitive identification of illegal drug use. Summary of the Invention

[0005] To overcome the deficiencies of the prior art, the present invention provides a SERS substrate based on mesoporous Au nanospheres, a preparation method thereof and an application thereof. Using Nα-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-lysine hydrochloride (Fmoc-D-Lys-OH·HCl) as a template agent, mesoporous 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 exclude interference from biological matrix macromolecules. This method is simple and time-saving, without complex sample pretreatment, and can achieve trace detection of MA and METH in urine.

[0006] The object of the present invention is achieved by the following technical solutions:

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

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

[0009] (2) Synthesis of mesoporous Au nanospheres: Under an ice bath at 2-4 °C, add an ascorbic acid solution to the template-metal precursor complex solution and stir evenly, carry out a 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 the SERS substrate: Drop the mesoporous Au nanosphere solution evenly on a substrate, dry it by baking, and then soak it in a mixed solution of acetone and acetic acid with a pH of 3-5 for 20-24 h to obtain the SERS substrate.

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

[0012] Preferably, the volume ratio of the Fmoc-D-Lys-OH·HCl solution, the chloroauric acid solution and deionized water in step (1) 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 revolutions per minute.

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

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

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

[0018] The second aspect of the present invention provides a SERS substrate prepared by the method described above, including a substrate and mesoporous Au nanospheres modifying the substrate. The mesoporous Au nanospheres present a bayberry-like morphology, with a diameter of 200 ± 25 nm, and have a mesoporous structure with three-dimensional radially open channels on the surface, and the pore diameter of the channels is 1.5 - 2.0 nm.

[0019] The third aspect of the present invention provides the application of the SERS substrate described above in the discrimination and detection of mandelic acid.

[0020] The fourth aspect of the present invention provides the application of the SERS substrate described above in the trace detection of methamphetamine.

[0021] The preparation method of the SERS substrate based on mesoporous Au nanospheres of the present invention, through a template-free and seed-free one-pot reduction system, under the regulation of the dynamic self-assembly of surfactant molecules, successfully constructs a mesoporous structure with three-dimensional radially open channels. This innovative design exhibits threefold synergistic advantages: First, the structure is precisely controllable and uniform, and the size and distribution density of the mesoporous channels can be precisely adjusted by the concentration of ascorbic acid solution and reaction kinetics, forming a high-density uniform and ordered "hot spot" region; Second, the selective molecular sieving function, the open pore network only allows the target analyte of specific molecules to efficiently penetrate into the interior of the particles through the pore size confinement effect, significantly enhancing the anti-interference ability of the substrate in complex biological matrices; Third, the in-situ chiral functionalization strategy, a chiral ligand of Fmoc-D-Lys-OH·HCl solution is synchronously introduced during the mesoporous growth process, and the directional self-assembly of chiral recognition sites is achieved by the synergistic action of π-π stacking and hydrogen bonding, which not only ensures the stability of the chiral surface conformation, but also avoids the problem of loss of active sites caused by traditional post-modification processes. This SERS substrate can realize the trace detection of MA and METH in urine, without being interfered by biological matrix macromolecules.

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

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

[0024] (2) The SERS substrate based on mesoporous Au nanospheres prepared by the method of the present invention 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, exclude the interference of biological matrix macromolecules, and have a significant nanoconfinement 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 has good anti-interference ability for the detection of METH in urine, and the lowest detection concentration of METH is 5 μM. Description of the Drawings

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

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

[0028] Figure 3 It is the N 1s spectra of 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 It is the characterization and analysis diagram of the mesoporous Au nanospheres prepared in Example 1. a and b are the low-magnification TEM images of the mesoporous Au nanospheres, and c and d are the high-resolution HAADF-STEM images of the mesoporous Au nanospheres.

[0030] Figure 5 It is 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 induced by Fmoc-D-Lys-OH·HCl and Fmoc-Lys-OH·HCl solutions.

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

[0032] Figure 7SERS enhancement and enantiomeric discrimination analysis of MA on the SERS substrate based on mesoporous Au nanospheres prepared in Example 1 after being soaked in acetone and acetic acid solutions for different soaking times (Au / D1 not soaked, Au / D2 soaked for 24 hours, Au / D3 soaked for one week); where a is the SERS spectral analysis of the SERS substrates of Au / D1, Au / D2, and Au / D3; b is the enantiomeric discrimination analysis of S-MA and R-MA on the SERS substrates with different cleaning times.

[0033] Figure 8 Analysis diagram of the selectivity effect 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 the selective SERS spectra of 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 respectively.

[0034] Figure 9 Enantiomeric discrimination analysis of MA on the SERS substrate based on mesoporous Au nanospheres prepared in Example 1. Where a is the Raman spectra of individual R-MA and S-MA, and b is the enantiomeric discrimination diagram of MA on the SERS substrate based on mesoporous Au nanospheres.

[0035] Figure 10 Detection limit analysis diagram of MA on the SERS substrate based on mesoporous Au nanospheres prepared in Example 1. Where 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 Selectivity analysis diagram of the SERS substrate based on mesoporous Au nanospheres prepared in Example 1 for MA enantiomers. Figure a is the SERS spectra of the substrate for different enantiomer (ee%) chiral recognition systems, and Figure b is the relationship between the difference in the relative intensity ratio of I 1002 / I 1022 and the ee value (%).

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

[0038] Figure 13 Detection analysis diagram of the urine samples of real drug addicts on the SERS substrate based on mesoporous Au nanospheres prepared in Example 1. Figure a is the SERS result of the substrate for urine sample detection, and Figure b is the comparison between the LC-MS detection result and the Raman result.

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

[0040] Figure 15 Chiral analysis diagrams of Au NPs of Comparative Example 2 and the SERS substrate modified with Fmoc-D-Lys-OH·HCl; Figure a shows the analysis results of MA for this SERS substrate, and Figure b shows the PCA analysis results. Detailed implementation manners

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

[0042] Example 1

[0043] A preparation method of a SERS substrate based on mesoporous Au nanospheres comprises the following steps:

[0044] (1) Prepare a template-metal precursor complex: Weigh 25 mg of Fmoc-D-Lys-OH·HCl (product number CAS number: 139262-23-0, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and dissolve it in 2.5 mL of ultrapure water. It needs to be dissolved by ultrasonic waves to obtain a 24.7 mM Fmoc-D-Lys-OH·HCl solution. Weigh 1 g of HAuCl4 (CAS number: 16903-35-8, purchased from Sinopharm Chemical Reagent Co., Ltd.) and dissolve it in 50 mL of ultrapure water to obtain a 50 mM HAuCl4 solution. Then, in a conical flask containing 17.5 mL of ultrapure water, add 2.5 mL of the Fmoc-D-Lys-OH·HCl solution and 0.5 mL of the 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 at a stirring speed of 1000 revolutions per minute.

[0045] (2) Synthesize mesoporous Au nanospheres: Weigh 0.176 g of ascorbic acid and dissolve it in 10 mL of deionized water. This step is carried out by shaking and vortexing to dissolve to obtain a 0.1 M ascorbic acid solution. Take 1 mL of the ascorbic acid solution and quickly add it to the template-metal precursor complex solution. The solution quickly changes from bright yellow to blue-green, and continue to react for 30 min. The color of the solution gradually deepens to obtain a mesoporous Au nanosphere solution. Centrifuge the mesoporous Au nanosphere solution and wash it with ultrapure water, repeat 1-2 times, the centrifugation time is 10 min, and the rotation speed is 5000 rpm. Then dissolve the obtained mesoporous Au nanospheres in 20 mL of ultrapure water to obtain a 2.5 mM mesoporous Au nanosphere solution. This step also must be carried out in an ice bath (4°C) and at a stirring speed of 1000 revolutions per minute.

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

[0047] The template-metal precursor complex was characterized by Cryo-EM technology, as Figure 1 shown. It can be seen that in this synthesis stage, the self-assembly process of nanospheres has begun, forming mesoporous nanospheres with certain size and structural characteristics. Molecules are orderly aggregated and self-assembled. This synthesis process depends on the micelle-directed arrangement to control the pore orientation and in-situ phase transformation to achieve mesoporous inheritance.

[0048] To deeply analyze the chemical state evolution of the template-metal precursor complex, XPS tests were carried out as Figure 2 shown. There are two characteristic peaks at 84.0 and 87.6 eV, which match with Au 4f 7 / 2 and Au 4f 5 / 2 respectively. The Au 4f peaks were further deconvolved to separate the signals from Au(I) and Au(0). Figure 3 Figure shows the N1s spectra of Fmoc-D-Lys-OH·HCl and the template-metal precursor complex. The amide bond of the Fmoc group is not directly involved in 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 forming a coordination bond (N→Au bond) with Au in HAuCl4. It shows 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+ to form Au(I) intermediates. Ascorbic acid preferentially reduces Au at the micelle-solution interface 3+ to form Au 3+ crystal nuclei, and the crystal nuclei grow epitaxially along the long axis of the micelles, inheriting the radial pore structure of the micelles. 0

[0049] The morphological characteristics and mesoporous structure of mesoporous Au nanospheres were systematically characterized based on low-magnification TEM and high-resolution HAADF-STEM, as Figure 4 shown. As Figure 4As shown in a and b, under low-magnification TEM observation, the mesoporous Au nanospheres all exhibit good monodispersity, with uniform spacing between the nanospheres. c and d are high-resolution HAADF-STEM images of the mesoporous Au nanospheres, further revealing the fine structure of the pores. The mesopore openings are relatively uniform and show a stacked pattern. The pore depth is about 50 - 80 nm, and clear lattice fringes can be seen on the inner wall. The main range of the pore size distribution is 1.5 - 2.0 nm. Mesopores of this size provide a rich pore structure inside the nanospheres, significantly increasing the number of hot spots inside the pores, thereby greatly improving the SERS activity of the material.

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

[0051] Comparative Example 1

[0052] The difference from Example 1 is only 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 Figure 1 b in the figure. It can be seen that the overall size of the L configuration is larger than that of the D configuration. Under the guidance of the D-configuration template, Fmoc-Lys molecules form a high-density self-assembly through π-π stacking and hydrogen bonding; while the L-configuration template has a lower ligand grafting density due to steric hindrance effects, resulting in a lower surface ligand content. This is also the reason why the effect of the L-configuration template is lower than that of the D-configuration template in the later MA enantiomer identification.

[0053] The chiral information of the mesoporous Au nanospheres in Example 1 and Comparative Example 1 was further analyzed by CD spectroscopy, as Figure 5 shown. It can be seen from a that the solutions obtained from Fmoc-D-Lys-OH·HCl and Fmoc-Lys-OH·HCl significantly show opposite chirality in the CD spectrum. It can be seen from b that the mesoporous Au nanospheres synthesized based on the template inducer show chiral information similar to 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 chiral gradient distribution of the local electric field. This special structure promotes the interaction between chiral molecules and the nanosphere surface, thereby enhancing the chiral signal.

[0054] To investigate the openness and selectivity of the pores of the mesoporous Au nanospheres prepared in Example 1 (Au-D) and Comparative Example 1 (Au-L), four different-sized model molecules with a common heme functional group were detected using the specific Raman fingerprint spectrum of the mesoporous Au nanospheres, 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. Solutions with certain concentrations were prepared respectively, mixed with the materials, and then centrifuged and washed thoroughly to remove unbound molecules. Subsequently, the materials were dropped onto a silicon wafer and their Raman spectra were collected. The experimental results are as Figure 8 shown. Only 1.2 nm hemin was completely retained and produced a strong Raman signal, and the 2.2 nm FITC-labeled recombinant protein was partially retained, confirming the size-selective confinement effect of the mesopores.

[0055] Comparative Example 2

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

[0057] Figure 14 In 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 conditions 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, because low temperature helps to control the crystal growth rate and reduce defects.

[0058] Comparative Example 3

[0059] A method for preparing a SERS substrate is as follows:

[0060] Prepare 40 nm Au NPs using the citrate reduction method and modify Fmoc-D-Lys-OH·HCl on their surface. Add 100 mL of chloroauric acid solution (0.01 wt% HAuCl4) to a round-bottom flask. Place the above solution on a stirrer, heat it to boiling and maintain the reflux state at the same time. When the solution boils, quickly add 1.0 mL of sodium citrate solution (1 wt%). Sodium citrate, as a reducing agent, can reduce chloroauric acid to Au NPs. Continue to boil the solution for 30 min to ensure the complete progress of the reaction. After the reaction is completed, let the solution cool naturally to room temperature to obtain a well-dispersed Au NPs solution. Subsequently, take 1-2 mL of the AuNPs solution and mix it with 1-2 mL of 10 mg / mL Fmoc-D-Lys-OH·HCl solution for modification for 30-60 min to obtain an Au nanosphere solution. Then, prepare the SERS substrate, following the steps in Example 1 (3) to obtain the SERS substrate.

[0061] Perform chiral analysis on the Au NPs of Comparative Example 3 and the SERS substrate modified with Fmoc-D-Lys-OH·HCl, but the results show that these substrates do not exhibit ideal chiral analysis effects. As Figure 15 shown, as can be seen from Figure 15 a in, smooth AuNPs can provide a certain SERS enhancement effect, but the signal at the position of the MA characteristic peak 1002 cm -1 is weak. Subsequently, 50 Raman spectra were collected respectively for PCA analysis, and the spectra showed a high degree of overlap (as shown in b). This is because of the lack of chiral features and symmetry breaking in its structure on the surface, and it is impossible to effectively distinguish R-MA and S-MA. Even after modifying Fmoc-D-Lys-OH·HCl on the surface of Au NPs, the chiral recognition ability has not been significantly improved. This indicates that it is difficult to achieve efficient chiral discrimination only by surface modification or smooth nanostructures.

[0062] Application Example 1

[0063] Use the SERS substrate based on mesoporous Au nanospheres of Example 1 for the enantiomeric discrimination detection of MA, and the steps are as follows:

[0064] Respectively take 2.5 μL of 10 mM R-MA and 10 mM S-MA aqueous solutions and drop them onto the SERS substrate, and let them dry naturally; use a 785 nm laser (power 0.1 mW) to collect Raman spectra (integration time 10 s).

[0065] Step (3) of the research: Effect of soaking time on chiral recognition of the SERS substrate. The following experiments were carried out on the SERS substrate respectively: not cleaned (Au / D1), cleaned for 24 h (Au / D2), cleaned for one week (Au / D3), and then SERS spectral analysis was performed, as Figure 7 shown. Among them, a is the SERS spectral analysis diagram of Au / D1, Au / D2, and Au / D3; b is the discrimination analysis diagram of S-MA and R-MA enantiomers by the SERS substrate cleaned for different aging times. It can be seen that the SERS substrate cleaned for 24 h shows the best SERS discrimination for MA enantiomers. Prolonging the cleaning time leads to signal disorder (carbonization) and loss of chiral function.

[0066] Discrimination of MA enantiomers by the SERS substrate based on mesoporous Au nanospheres is as Figure 9 shown. a is the Raman spectra of R-MA and S-MA alone, and b is the discrimination diagram of MA enantiomers by the SERS substrate based on mesoporous Au nanospheres. It can be seen that new characteristic peaks appear at 618 cm -1 , 826 cm -1 , 858 cm -1 , 1002 cm -1 , 1174 cm -1 , 1197 cm -1 , 1537 cm -1 and 1593 cm -1 , which are consistent with the characteristic Raman bands of MA. Based on mesoporous Au nanospheres as the SERS substrate, different action modes are generated for R-MA and S-MA enantiomers. There are obvious differences in the relative intensities of the two spectral bands at 1002 cm -1 and 1593 cm -1 . When S-MA participates in the chiral recognition system, the spectral band intensities at 1002 cm -1 and 1593 cm -1 are much higher than those 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 respectively, and SERS analysis was carried out, as Figure 10 shown. Figure a is the sensitivity analysis of R-MA, and Figure b is the sensitivity analysis of S-MA. As the concentration of MA increases, the intensity of the SERS signal also increases, indicating that the material has good concentration dependence. Especially for S-MA, even at a low concentration of 0.1 mM, weak but clear MA characteristic signals can still be detected, indicating that the SERS substrate based on mesoporous Au nanospheres has high detection sensitivity.

[0068] To deeply 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. As shown in Figure 11 the figure, a is the SERS spectrum of the chiral recognition system with different enantiomers (ee%), and b is the I 1002 / I 1022 relationship between the difference in relative intensity ratios and the ee value (%). It can be seen that as the concentration of S-MA in the MA enantiomer solution increases, the intensity of the main Raman peak at 1002 cm -1 gradually increases. Taking the relative intensity ratio of (I 1002 / I 1022 ) as the chiral selectivity internal standard, this ratio gradually increases as the ee% value of S-MA increases. This indicates 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 trace detection of METH in urine. The steps are as follows:

[0071] Take 1 mL of urine from drug addicts, add 4 mL of ultrapure water for dilution, vortex and mix evenly. Take 2.5 μL of the diluted urine and drop it onto the SERS substrate of Example 1, let it stand for 1 min for adsorption, use a 785 nm laser (power 0.1 mW), and collect the Raman spectrum (integration time 10 s).

[0072] To explore the anti-interference ability of the SERS substrate based on mesoporous Au nanospheres for real biological samples, we tested the interfering components such as urea, uric acid, and glucose in urine. The experimental results are as shown in Figure 12 the figure. Even after adding 100 mM of the interfering components to the substrate, the spectrum did not show the characteristic signals of the interfering components. Affected by the nanoconfinement of the material, these molecules did not enter the pores and interact with the substrate.

[0073] The concentration of METH in the same urine sample was independently detected using LC-MS technology, and the results of the two were compared. As shown in Figure 13As shown, a is the detection result graph of METH concentration by Raman substrate, b is the detection result graph of METH concentration by LC-MS. The deviation between SERS and LC-MS results is small, which confirms its clinical-level detection reliability in complex biological matrices, high sensitivity and high selectivity analysis of METH for drug abuse, and the lowest detection concentration is 5 μM. The mesoporous channels of the nanospheres allow METH molecules smaller than the pores to selectively enter and exclude proteins or macromolecules larger than the cut-off value, enabling direct detection of small molecules in complex biological samples and avoiding cumbersome sample pretreatment.

[0074] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the inventive concept, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A preparation method of a SERS substrate based on mesoporous Au nanospheres, characterized in that, It includes the following steps: (1) Preparation of a template-metal precursor complex: Under an ice bath at 2-4 °C, a solution of Nα-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-lysine hydrochloride and a solution of chloroauric acid are mixed, and then deionized water is added and stirred evenly to form a template-metal precursor complex; (2) Synthesis of mesoporous Au nanospheres: Under an ice bath at 2-4 °C, ascorbic acid solution is added to the template-metal precursor complex and stirred evenly, and a co-reduction reaction is carried out for 20-40 min, followed by centrifugation and washing, and then redispersed in ultrapure water to obtain a 2-3 mM mesoporous Au nanosphere solution; (3) Preparation of a SERS substrate: The mesoporous Au nanosphere solution is evenly dropped on a substrate, dried by baking, and then soaked in a mixed solution of acetone and acetic acid for 20-24 h 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, the 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, In step (2), the concentration of the ascorbic acid solution 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, wherein In step (2), the rotation speed of the centrifugation is 4000-6000 rpm, and the time is 8-15 min.

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

7. The preparation method according to claim 1, characterized in that, In step (3), the pH of the mixed solution 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 preparation method according to any one of claims 1 to 7, characterized in that, It includes a substrate and mesoporous Au nanospheres modifying the substrate. The mesoporous Au nanospheres exhibit a bayberry-like morphology, with a diameter of 200±25 nm, and have a mesoporous structure with three-dimensional radially open pores on the surface, and the pore diameter of the pores is 1.5-2.0 nm.

9. Application of the SERS substrate according to claim 8 in the identification and detection of mandelic acid.

10. Application of the SERS substrate according to claim 8 in the trace detection of methamphetamine.

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