Enantiomer selective surface enhanced Raman scattering substrate and preparation method and application thereof

Abstract

The invention belongs to the technical field of SERS (Surface Enhanced Raman Scattering) detection, and relates to an enantioselective surface enhanced Raman scattering substrate as well as a preparation method and application thereof. Chiral spiral gold nanorods (L-HAuNRs) are synthesized through chiral molecule induction, self-assembly of the chiral gold nanorods is promoted by adjusting the PH of a solution and adding a thiol ligand, then the chiral gold nanorods are coupled with a deposited gold nanofilm (AuF), and the enantiomer selective surface enhanced Raman scattering substrate based on the chiral gold rod coupled gold film is prepared. According to the substrate, the chiral optical activity of L-HAuNRs and the strong adsorption capacity of AuF are combined, the L-HAuNRs and the AuF are coupled to localize a light field in a nano gap cavity, and a Raman signal is remarkably amplified through a localized strong electric field. The substrate shows excellent SERS (Surface Enhanced Raman Scattering) identification capability on a phenylalanine enantiomer and a chiral metabolism drug levofloxacin in human sweat, and has high sensitivity and good stability.

Description

Technical Field

[0001] This invention belongs to the field of SERS detection technology, specifically relating to an enantioselective surface-enhanced Raman scattering substrate, its preparation method, and its application. Background Technology

[0002] Chirality, the property of an object not being able to perfectly superimpose its mirror image, is a crucial foundation for the origin and evolution of life. The human body possesses a unique chiral environment; for example, the natural amino acids that make up the basic building blocks of proteins are primarily in the L-configuration, while the monosaccharides that form the backbone of nucleic acids (such as DNA and RNA) are mostly in the D-configuration. Enantiomers of human metabolites are key biomarkers for the diagnosis and monitoring of diseases such as cancer, neurological disorders, and diabetes. Furthermore, in a chiral biological environment, enantiomers of drug molecules often exhibit significantly different pharmacological effects and toxicological characteristics. Therefore, the identification and analysis of chiral enantiomers have significant applications in biomedicine, clinical diagnostics, and drug development.

[0003] Surface-enhanced Raman scattering (SERS) is a powerful analytical technique that enables precise molecular identification by characterizing the vibrational fingerprints of trace analytes. Chiral plasmon nanostructures possess the ability to enhance electromagnetic fields and modulate chiral optical interactions at the nanoscale, allowing for selective resonant coupling with enantiomers and thus enabling enantiomer identification via the SERS-chiral anisotropy (SERS-ChA) effect. However, the SERS-ChA activity of current single-component plasmon nanostructures remains relatively weak, posing challenges to the precise identification and quantitative detection of trace analytes. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides an enantioselective surface-enhanced Raman scattering substrate, which is composed of self-assembled chiral helical gold nanorods (L-HAuNRs) coupled with gold nanofilms (AuF). By utilizing the chiral properties of L-HAuNRs and the strong adsorption capacity of AuF, as well as the local electromagnetic field and chiral optical response generated after the coupling of the two, ultrasensitive recognition of chiral molecules can be achieved, and the dynamic metabolic pattern of the chiral metabolic drug levofloxacin in human sweat can be accurately captured.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides an enantioselective surface-enhanced Raman scattering substrate, the substrate being composed of self-assembled chiral gold nanorods (L-HAuNRs) coupled with a gold nanofilm (AuF); the chiral gold nanorods have a helical morphology with an average rod length of 120–160 nm, a pitch length of 20–25 nm, a pitch depth of 8–12 nm, and exhibit distinct transverse and longitudinal plasmon resonance characteristic peaks at 520–530 nm and 750–780 nm, respectively; the gold nanofilm has a thickness of 20–70 nm.

[0006] A second aspect of the present invention also provides a method for preparing the enantioselective surface-enhanced Raman scattering substrate described in the first aspect, the method comprising the following steps: S1. Mix chloroauric acid solution with CTAB solution to form a bright yellow solution, then add sodium borohydride solution treated in an ice bath. After vigorous stirring, the solution turns brownish-yellow and is left to stand at room temperature to age to obtain gold seed solution. S2. Dissolve CTAB and sodium oleate (NaOL) in water, then add silver nitrate solution and chloroauric acid solution in sequence. After stirring, add hydrochloric acid to adjust the pH value to obtain the growth solution. S3. Using the seed-mediated method, the gold seed solution from step S1 and ascorbic acid solution are added to the growth solution from step S2. After stirring, standing and centrifugation purification, a gold nanorod solution is obtained. S4. The gold nanorod solution was redispersed in CTAB solution, then L-cysteine ​​solution was added. After standing, silver nitrate solution was added and stirred until well mixed. Chloroauric acid solution and ascorbic acid solution were added in sequence. The mixture was then heated in a water bath. The product was centrifuged to remove the supernatant and redispersed in an aqueous solution. Sodium hydroxide was added to adjust the overall pH of the solution. Then 3-mercaptopropionic acid (MPA) solution was added and stirred until well mixed. The mixture was heated in a water bath for a period of time to obtain a self-assembled L-type chiral helical gold nanorod (L-HAuNRs) solution. S5. Sputter deposit gold nanofilms on the substrate, then ultrasonically clean them with acetone and ethanol in sequence, and dry them with nitrogen to obtain clean gold nanofilms (AuF). S6. Drop the self-assembled L-shaped chiral helical gold nanorod solution onto the surface of the gold nanofilm. After drying in a dust-proof environment, the chiral gold nanorod coupled gold film (L-HAuNRs / AuF) structure is obtained, which is an enantioselective surface-enhanced Raman scattering substrate.

[0007] Preferably, in S1, the molar concentration of the chloroauric acid solution is 0.3–0.6 mM, the molar concentration of the CTAB solution is 0.1–0.3 M, and the molar concentration of the sodium borohydride solution is 10–15 mM. The volume ratio of the chloroauric acid solution, CTAB solution, and sodium borohydride solution is 1:1:0.5–0.7. The stirring speed is 1000–1400 rpm, the stirring time is 1–3 minutes, and the aging time is 2–3 hours.

[0008] Preferably, in step S2, the mass-to-volume ratio of CTAB to water is 3.5–4.5 g: 125 mL, and the mass ratio of CTAB to sodium oleate is 5–6:1; the molar concentration of the silver nitrate solution is 3–5 mM, the molar concentration of the chloroauric acid solution is 1–2 mM, the volume ratio of the silver nitrate solution to the chloroauric acid solution is 12–15: 123–127 mL, and the volume ratio of the chloroauric acid solution to water is 1:1; the stirring speed is 500–800 rpm, and the stirring time is 1.5–2 hours; the mass fraction of the hydrochloric acid is 37%, and the volume ratio of the hydrochloric acid to water is 0.9–1.2: 125.

[0009] Preferably, in step S3, the volume ratio of the growth solution, the gold seed solution, and the ascorbic acid solution is 260–265: 0.2–0.3: 0.55–0.7, and the molar concentration of the ascorbic acid solution is 0.06–0.07 M; the stirring speed is 800–1200 rpm, the stirring time is 1–3 min; the settling time is 8–12 h; and the centrifugation speed is 6000–7500 rpm, the centrifugation time is 10–20 min.

[0010] Preferably, in step S4, the molar concentration of the CTAB solution is 9–12 mM, the molar concentration of the L-cysteine ​​solution is 1–6 mM, the molar concentration of the silver nitrate solution is 1–2 mM, the molar concentration of the chloroauric acid solution is 4–6 mM, and the molar concentration of the ascorbic acid solution is 0.1–0.2 M; the volume ratio of the CTAB solution, L-cysteine ​​solution, silver nitrate solution, chloroauric acid solution, and ascorbic acid solution is 45–50:1:0.3–0.5:1–3:0.3–0.5; the standing time is 10–15 h; the stirring speed is 100–250 rpm, and the stirring time is 10–30 min; the heating temperature is 60–80℃, and the time is 1.5–2 h; the centrifugation speed is 5000–7000 rpm, and the time is 5–10 min. min; after redispersing and adding sodium hydroxide, adjust the overall pH of the solution to 8-10, add 3-mercaptopropionic acid to gold nanorods in a molar ratio of 1-3:1, and heat in a water bath at 35-50 ℃ for 15-90 min.

[0011] Preferably, in step S5, a gold nanofilm is deposited on the substrate by ion sputtering for a time of 1 to 5 minutes; the substrate includes a silicon wafer or a polydimethylsiloxane (PDMS) substrate.

[0012] Preferably, in step S6, the concentration of the self-assembled L-shaped chiral helical gold nanorod solution is 0.1–1 mg / mL; the drying is natural drying for 24–48 h.

[0013] The third aspect of this invention also provides the application of the enantioselective surface-enhanced Raman scattering substrate described in the first aspect in detecting and identifying phenylalanine enantiomeric molecules or in real-time monitoring of the chiral metabolite drug levofloxacin in human sweat.

[0014] Preferably, the method for detecting and identifying enantiomers of phenylalanine molecules specifically involves: mixing the enantiomerically selective surface-enhanced Raman scattering substrate described in the first aspect with solutions of levorotatory and dextrorotatory phenylalanine molecules, respectively; after mixing and adsorption for more than 2 hours, drying the mixture; and performing Raman spectroscopy measurements using a confocal Raman spectrometer, wherein the excitation wavelength of the Raman spectrum is 532 nm and the power is 1–5 mW; wherein the SERS ratio of levorotatory phenylalanine to dextrorotatory phenylalanine is stably maintained at a difference of more than three times; The specific method for monitoring the chiral metabolite levofloxacin in human sweat is as follows: First, levofloxacin (L-vfx) is dissolved in artificial sweat to prepare a series of solutions with different concentrations. The substrate described in the first aspect is mixed with the solution and adsorbed for more than 2 hours, then dried. Raman spectroscopy is performed to measure the concentration and a standard curve of the intensity-concentration of the characteristic Raman peak of L-vfx is plotted. Then, the human sweat to be tested is collected, dropped onto the substrate described in the first aspect, and dried. Raman spectroscopy is also performed to measure the concentration. Finally, the corresponding concentration of levofloxacin (L-vfx) is obtained by combining the standard curve. The excitation wavelength of the Raman spectroscopy is 532 nm, and the power is 1~5 milliwatts.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention provides an enantioselective surface-enhanced Raman scattering (SERS) substrate, which is composed of self-assembled chiral helical gold nanorods (L-HAuNRs) coupled with a gold nanofilm (AuF). The preparation method is as follows: First, helical gold nanorods (L-HAuNRs) with strong chiral optical activity are grown using chiral molecule induction. Then, by adjusting the solution pH and adding a thiol ligand, self-assembled chiral helical gold nanorods are obtained. Subsequently, the self-assembled L-HAuNRs are coupled with AuF to finally prepare a chiral plasmon surface-enhanced Raman scattering substrate, which can achieve ultrasensitive detection of chiral molecules. On the one hand, this substrate fully utilizes the roughness of the gold nanofilm, which can effectively adsorb target molecules; on the other hand, the chiral properties of the helical gold nanorods can selectively resonate with enantiomeric molecules. On the other hand, the optimized chiral helical gold nanorod / gold nanofilm (HAuNRs / AuF) substrate can localize the light field within the nano-interval cavity, significantly enhancing the Raman signal through a localized strong electric field. This results in excellent SERS discrimination of phenylalanine enantiomers, with a detection limit as low as 10⁻⁶. -8 M exhibits high detection sensitivity and stability. Simultaneously, this substrate can accurately capture the dynamic metabolic patterns of levofloxacin, a chiral metabolite in human sweat, providing a new approach for developing advanced biosensor chips for detecting disease-related chiral molecules, and showing great potential in diagnostics, prognosis, and personalized medicine. Attached Figure Description

[0016] Figure 1 Transmission electron microscopy image of self-assembled chiral helical gold nanorods (L-HAuNRs); Figure 2 The absorption spectra of AuNRs and L-HAuNRs are shown. Figure 3 The circular dichroism spectra of AuNRs and L-HAuNRs; Figure 4 Raman spectra of L-Phe molecules at different concentrations were detected on an L-HAuNRs / AuF Raman substrate; Figure 5 Raman spectra of D-Phe molecules at different concentrations were detected on an L-HAuNRs / AuF Raman substrate.

[0017] Figure 6 Levofloxacin at 1402 cm⁻¹ on an L-HAuNRs / AuF substrate. -1 Linear relationship between characteristic peak intensity and concentration (0.1-20 μM); Figure 7 The graph shows the metabolic curve of levofloxacin concentration in sweat over time on an L-HAuNRs / AuF substrate. Detailed Implementation

[0018] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0019] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0020] Example 1: Preparation of an enantioselective surface-enhanced Raman scattering substrate (chiral gold nanorods L-HAuNRs coupled with gold nanofilm AuF) (1) Mix 5 mL of chloroauric acid solution (0.5 mM) with 5 mL of CTAB solution (0.2 M). After mixing, the solution is bright yellow. Then add 600 μL of sodium borohydride solution (10 mM) that has been treated in an ice bath. After stirring vigorously (1200 rpm) for 2 minutes, let the solution stand at room temperature for 3 hours to obtain a brownish-yellow gold seed solution. (2) Weigh 3.5 g CTAB and 0.617 g NaOL powder into 125 mL pure water, dissolve them completely, add 12 mL silver nitrate solution (4 mM), let stand for 15 minutes, then add 125 mL chloroauric acid solution (1 mM), stir continuously at 700 rpm for 90 minutes, and then add 0.9 mL hydrochloric acid (37% mass fraction) to adjust the pH value to obtain the growth solution; (3) Using the seed-mediated method, 0.25 mL of seed liquid and 0.625 mL of ascorbic acid solution (0.064 M) were added to the above growth solution. After stirring at 1000 rpm for 2 minutes, the solution was left to stand overnight to grow, and gold nanorod (AuNRs) solution was obtained. The obtained AuNRs solution was centrifuged twice (7000 rpm, 10 min) to remove high concentrations of CTAB. (4) The centrifuged purified AuNRs 0.6 mg was redispersed in 0.01 M CTAB solution (49 mL) to maintain good dispersibility, and then 1 mL of L-cysteine ​​(L-Cys) aqueous solution (6 mM) was added and allowed to stand for 12 h to form a homogeneous solution. (5) Add 0.35 mL of silver nitrate solution (1 mM) to the above solution and stir at 100 rpm for 15 min; (6) Add 1.3 mL of chloroauric acid solution (5 mM) and 0.33 mL of ascorbic acid solution (0.1 M) to the above solution, mix thoroughly, heat in a 70°C water bath for 2 h, and after the solution cools, centrifuge twice at 6500 rpm for 5 min to remove the supernatant, and redisperse with ultrapure water (0.2 mg / mL) to obtain L-HAuNRs solution; add NaOH to L-HAuNRs solution to make the overall pH of the solution 8, then add 100 μL of 0.1 M MPA solution, heat in a 40°C water bath for 45 min to obtain self-assembled L-HAuNRs solution; (7) Using gold (Au) as the target material, gold nanofilm (Aufilm, AuF) was deposited on a silicon wafer (1cm×1cm) using an ion sputtering instrument. The sputtering time was 4 min, and the thickness of the obtained AuF was 60 nm. The AuF was then ultrasonically cleaned with acetone and anhydrous ethanol in sequence. The ultrasonic cleaning was performed at 100 mW power for 3 min, and the AuF was dried with nitrogen gas to obtain clean AuF. (8) Take 20 μL of self-assembled L-HAuNRs solution with a concentration of 0.5 mg / mL and drop it onto the AuF surface. After drying naturally in a dust-proof environment for 36 h, a chiral gold nanorod coupled gold film (L-HAuNRs / AuF) structure is obtained.

[0021] like Figure 1 As shown, the chiral gold nanorods exhibit a helical morphology with an average rod length of 140 nm, a pitch length of 22 nm, and a pitch depth of 11 nm. They also display distinct transverse and longitudinal plasmon resonance characteristic peaks at approximately 538 nm and 768 nm, respectively (e.g., ...). Figure 2 As shown); Figure 3 As shown, AuNRs do not have obvious circular dichroism signals, while the CD spectrum of L-HAuNRs shows a strong negative peak at a short wavelength (541 nm) and a weak positive peak at a long wavelength (750 nm).

[0022] Example 2: Ability of enantioselective surface-enhanced Raman scattering substrates (L-HAuNRs / AuF) to identify chiral phenylalanine molecules The L-HAuNRs / AuF substrate prepared in Example 1 was immersed in different concentrations (10). -8 ~10 -3 The mixture was placed in a solution of L-phenylalanine (L-Phe) or D-phenylalanine (D-Phe) for 3 hours for adsorption, then dried and subjected to Raman spectroscopy using a confocal Raman spectrometer with an excitation wavelength of 532 nm and a power of 1 mW.

[0023] The results showed that L-HAuNRs / AuF could detect the SERS ratio of L-Phe and D-Phe consistently maintained a difference of more than three times, and the detection limit for L-Phe was as low as 10. -8 The detection limit for D-Phe is as low as 10 moles per liter. -7 moles per liter (e.g.) Figure 4 , 5 (As shown).

[0024] Example 3: Application of enantioselective surface-enhanced Raman scattering substrate (L-HAuNRs / AuF) in real-time monitoring of chiral metabolized drug levofloxacin in human sweat First, levofloxacin (L-vfx) was dissolved in artificial sweat (sodium chloride NaCl: 2.0% (20 g / L), ammonium chloride NH4Cl: 1.75% (17.5 g / L), urea CH4N2O: 0.5% (5 g / L), lactic acid C3H6O3: 1.5% (15 g / L), acetic acid CH3COOH: 0.25% (2.5 g / L; the remainder is water, accounting for approximately 94%) to prepare a series of solutions of different concentrations (0.1-20 μM). The L-HAuNRs / AuF substrate prepared in Example 1 was immersed in the solution for 3 hours for adsorption and then dried. Raman spectroscopy was performed using a confocal Raman spectrometer. The results showed that L-vfx was spectrally active at 1402 cm⁻¹. -1 The characteristic peak intensity at a given location shows a linear correlation with the concentration (e.g., Figure 6 As shown), the Raman spectroscopy excitation wavelength was 532 nm, and the power was 1 mW. Next, 1 mL of human sweat secreted by volunteers after ingesting 500 mg of levofloxacin tablets was collected and dropped onto the L-HAuNRs / AuF substrate prepared in Example 1. After drying at room temperature, Raman spectroscopy was performed using a confocal Raman spectrometer, with the excitation wavelength and power being the same as above. Finally, the corresponding L-vfx concentrations at different metabolic times (e.g., [data missing]) were obtained based on the intensity-concentration linear curve. Figure 7 As shown in the figure, L-HAuNRs / AuF can accurately capture the dynamic metabolic patterns of L-vfx in an individual.

[0025] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. An enantioselective surface-enhanced Raman scattering substrate, characterized in that, The substrate is composed of self-assembled chiral gold nanorods coupled with gold nanofilms; the chiral gold nanorods have a helical morphology with an average rod length of 120–160 nm, a pitch length of 20–25 nm, a pitch depth of 8–12 nm, and exhibit distinct transverse and longitudinal plasmon resonance characteristic peaks at 520–530 nm and 750–780 nm, respectively; the gold nanofilm has a thickness of 20–70 nm.

2. The method for preparing the enantioselective surface-enhanced Raman scattering substrate according to claim 1, characterized in that, Includes the following steps: S1. Mix chloroauric acid solution with CTAB solution to form a bright yellow solution, then add sodium borohydride solution treated in an ice bath. After vigorous stirring, the solution turns brownish-yellow and is left to stand at room temperature to age to obtain gold seed solution. S2. Dissolve CTAB and sodium oleate in water, then add silver nitrate solution and chloroauric acid solution in sequence. After stirring, add hydrochloric acid to adjust the pH value to obtain the growth solution. S3. Using the seed-mediated method, the gold seed solution from step S1 and ascorbic acid solution are added to the growth solution from step S2. After stirring, standing and centrifugation purification, a gold nanorod solution is obtained. S4. The gold nanorod solution was redispersed in CTAB solution, then L-cysteine ​​solution was added. After standing, silver nitrate solution was added and stirred until well mixed. Chloroauric acid solution and ascorbic acid solution were added in sequence. The mixed solution was then heated in a water bath. The product was centrifuged to remove the supernatant and redispersed in an aqueous solution. Sodium hydroxide was added to adjust the overall pH of the solution. Then 3-mercaptopropionic acid solution was added and stirred until well mixed. The solution was heated in a water bath for a period of time to obtain a self-assembled L-type chiral helical gold nanorod solution. S5. Sputter deposit gold nanofilms on the substrate, then ultrasonically clean them with acetone and ethanol in sequence, and dry them with nitrogen to obtain clean gold nanofilms. S6. Drop the self-assembled L-shaped chiral helical gold nanorod solution onto the surface of the gold nanofilm. After drying in a dust-proof environment, the chiral gold nanorod coupled gold film structure is obtained, which is an enantioselective surface-enhanced Raman scattering substrate.

3. The method for preparing an enantioselective surface-enhanced Raman scattering substrate according to claim 2, characterized in that, In S1, the molar concentration of the chloroauric acid solution is 0.3–0.6 mM, the molar concentration of the CTAB solution is 0.1–0.3 M, and the molar concentration of the sodium borohydride solution is 10–15 mM. The volume ratio of the chloroauric acid solution, CTAB solution, and sodium borohydride solution is 1:1:0.5–0.

7. The stirring speed is 1000–1400 rpm, the stirring time is 1–3 minutes, and the aging time is 2–3 hours.

4. The method for preparing an enantioselective surface-enhanced Raman scattering substrate according to claim 2, characterized in that, In S2, the mass-to-volume ratio of CTAB to water is 3.5–4.5 g: 125 mL, and the mass ratio of CTAB to sodium oleate is 5–6:1; the molar concentration of the silver nitrate solution is 3–5 mM, the molar concentration of the chloroauric acid solution is 1–2 mM, the volume ratio of the silver nitrate solution to the chloroauric acid solution is 12–15: 123–127 mL, and the volume ratio of the chloroauric acid solution to water is 1:1; the stirring speed is 500–800 rpm, and the stirring time is 1.5–2 hours; the mass fraction of the hydrochloric acid is 37%, and the volume ratio of the hydrochloric acid to water is 0.9–1.2:

125.

5. The method for preparing an enantioselective surface-enhanced Raman scattering substrate according to claim 2, characterized in that, In step S3, the volume ratio of the growth solution, gold seed solution, and ascorbic acid solution is 260–265:0.2–0.3:0.55–0.7, and the molar concentration of the ascorbic acid solution is 0.06–0.07 M; the stirring speed is 800–1200 rpm, and the stirring time is 1–3 min; the standing time is 8–12 h; and the centrifugation speed is 6000–7500 rpm, and the centrifugation time is 10–20 min.

6. The method for preparing an enantioselective surface-enhanced Raman scattering substrate according to claim 2, characterized in that, In step S4, the molar concentration of the CTAB solution is 9–12 mM, the molar concentration of the L-cysteine ​​solution is 1–6 mM, the molar concentration of the silver nitrate solution is 1–2 mM, the molar concentration of the chloroauric acid solution is 4–6 mM, and the molar concentration of the ascorbic acid solution is 0.1–0.2 M; the volume ratio of the CTAB solution, L-cysteine ​​solution, silver nitrate solution, chloroauric acid solution, and ascorbic acid solution is 45–50:1:0.3–0.5:1–3:0.3–0.5; the standing time is 10–15 h; the stirring speed is 100–250 rpm, and the stirring time is 10–30 min; the heating temperature is 60–80℃, and the time is 1.5–2 h; the centrifugation speed is 5000–7000 rpm, and the time is 5–10 min. min; after redispersing and adding sodium hydroxide, adjust the overall pH of the solution to 8-10, add 3-mercaptopropionic acid to gold nanorods in a molar ratio of 1-3:1, and heat in a water bath at 35-50 ℃ for 15-90 min.

7. The method for preparing an enantioselective surface-enhanced Raman scattering substrate according to claim 2, characterized in that, In S5, gold nanofilms are deposited on the substrate by ion sputtering for 1–5 min.

8. The method for preparing an enantioselective surface-enhanced Raman scattering substrate according to claim 2, characterized in that, In S6, the concentration of the self-assembled L-type chiral helical gold nanorod solution is 0.1–1 mg / mL; the drying is natural drying for 24–48 h.

9. The application of the enantioselective surface-enhanced Raman scattering substrate of claim 1 in detecting and identifying phenylalanine enantiomeric molecules or in real-time monitoring of the chiral metabolite drug levofloxacin in human sweat.

10. The application according to claim 9, characterized in that, The method for detecting and identifying enantiomers of phenylalanine molecules is as follows: the enantiomer-selective surface-enhanced Raman scattering substrate described in claim 1 is mixed with solutions of levorotatory and dextrorotatory phenylalanine molecules, respectively. After mixing and adsorption for more than 2 hours, the mixture is dried, and Raman spectroscopy is performed using a confocal Raman spectrometer. The excitation wavelength of the Raman spectrum is 532 nm, and the power is 1-5 mW. The SERS ratio of levorotatory phenylalanine to dextrorotatory phenylalanine is stably maintained at a difference of more than three times. The specific method for monitoring the chiral metabolite levofloxacin in human sweat is as follows: First, levofloxacin (L-vfx) is dissolved in artificial sweat to prepare a series of solutions with different concentrations. The substrate described in claim 1 is mixed with the solution and adsorbed for more than 2 hours, then dried. Raman spectroscopy is performed to measure the concentration and a standard curve of the characteristic Raman peak of L-vfx is plotted. Then, the human sweat to be tested is collected, dropped onto the substrate described in claim 1, and dried. Raman spectroscopy is also used for measurement. Finally, the corresponding concentration of levofloxacin (L-vfx) is obtained by combining the standard curve. The excitation wavelength of the Raman spectroscopy is 532 nm, and the power is 1~5 milliwatts.

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