Multidimensional plasmonic coupling system based on Ag@PVP and Au@Ag NCs for surface-enhanced Raman spectroscopy (SERS) detection of gentamicin

Abstract

The multi-dimensional plasmonic coupling system based on Ag@PVP and Au@Ag NCs for surface-enhanced Raman spectroscopy (SERS) detection of gentamicin relates to the field of analytical detection, and adopts hydrothermal synthesis to prepare Ag@PVP core-shell nanoparticles and Au@Ag core-shell cubes (Au@Ag NCs). Ag@PVP is induced to agglomerate by gentamicin, and then Au@Ag NCs are introduced to change the electromagnetic field of the nanoparticle agglomeration, so as to change the Raman characteristic peaks of gentamicin and distinguish it from its homologues. Under the optimized experimental conditions, the linear detection range of gentamicin is 10 ‑5 ~10 ‑9 M, the detection limit is 6.23 × 10 ‑10 M, and the relative standard deviation of SERS intensity is less than 10%. The method has the advantages of simple operation, high accuracy and sensitivity, and does not need complex operation technology, and meets the requirements of large quantities and rapid analysis and detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection, and in particular to a multi-dimensional plasmonic coupling system based on Ag@PVP and Au@Ag NCs for surface-enhanced Raman spectroscopy (SERS) detection of gentamicin. BACKGROUND

[0002] Gentamicin (Gen) is an aminoglycoside antibiotic. Due to its molecular structure with a large number of positive charges, it exists in the form of the original drug in the body and is difficult to be metabolically degraded. Its main excretion pathway is through the kidneys, but a small part is enriched in the epithelial cells of the proximal tubules of the kidneys. This enrichment can cause damage to kidney function and slow down drug excretion, forming a vicious cycle. Due to the inability of the drug to be effectively excreted in the body, nephrotoxicity is produced, further causing ototoxicity, which can cause damage to the nervous function such as dizziness, nausea and nystagmus. In severe cases, it can cause allergic reactions such as shock, red rash, and even death. Therefore, in order to protect food safety, the maximum residue limit of gentamicin in animal food is 200 μg / kg in GB 31650-2019 "National Food Safety Standard Maximum Residue Limits of Veterinary Drugs in Food". Therefore, it is particularly important to develop a high-sensitivity and high-selectivity detection method for gentamicin drug residues. However, there are great challenges in achieving this goal, mainly because gentamicin lacks a chromophore or a fluorescent group, which requires derivatization.

[0003] Currently, the detection methods for gentamicin mainly include high-performance liquid chromatography, fluorescence immunoassay, ultraviolet spectrophotometry, liquid chromatography-mass spectrometry, and microbiological method. The column in high-performance liquid chromatography requires very high pretreatment of the sample, which consumes a large amount of liquid and reagent consumables, and is easy to block the column and cause economic loss; the fluorescence detection method requires pre-derivatization, which is complicated and time-consuming, and incomplete derivatization can also reduce the sensitivity of detection; liquid chromatography-mass spectrometry is more convenient than ultraviolet and fluorescence detection, does not require a derivatization step, and has significantly higher sensitivity than microbiological methods, but it requires higher professional skills of the operator and is more expensive in equipment cost, so it is not suitable for rapid screening and popularization of large quantities of samples. SUMMARY

[0004] The purpose of the present application is to solve the above-mentioned problems in the prior art, to provide a simple, rapid and reliable SERS detection method for gentamicin, and to prepare Ag@PVP and Au@Ag NCs as SERS substrates for quantitative analysis of gentamicin. Under the optimized volume ratio and pH conditions, qualitative and quantitative analysis of gentamicin is achieved. The linear detection range of gentamicin is 10 -5 ~ 10 -9M, the detection limit is 6.23*10 -10 M, the relative standard deviation of SERS intensity is less than 10%. The method has the advantages of low cost, controllability, high detection efficiency, simple operation, high accuracy and sensitivity, and does not need complex operation technology, and can meet the requirements of large batch and rapid analysis and detection, and can be popularized and applied.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0006] The multi-dimensional plasmonic coupling system based on Ag@PVP and Au@Ag NCs is used for surface enhanced Raman spectroscopy (SERS) detection of gentamicin, comprising the following steps:

[0007] 1) preparing Ag@PVP core-shell nanoparticles;

[0008] 2) preparing Au@Ag NCs sol;

[0009] 3) mixing the gentamicin sample solution with Ag@PVP core-shell nanoparticles, then adding Au@Ag NCs sol, and distinguishing from other homologues by the Raman characteristic peak of gentamicin;

[0010] 4) quantitative analysis and detection of gentamicin.

[0011] In step 1), silver sol is prepared by sodium citrate reduction method, and then PVP is added to the silver sol to prepare the Ag@PVP core-shell nanoparticles.

[0012] In step 2), gold seed solution and growth solution are synthesized respectively, the two solutions are mixed and then placed to obtain gold growth solution, then the gold growth solution is reacted with CTAC solution, and then silver nitrate solution and ascorbic acid-CTAC solution are injected at the same time to prepare the Au@Ag NCs sol.

[0013] The gentamicin sample solution is mixed with Ag@PVP core-shell nanoparticles, so that gentamicin induces the aggregation of Ag@PVP, and the Raman characteristic peak of gentamicin appears at 948 cm -1 ; after adding Au@Ag NCs sol, Au@Ag NCs regulates the surface plasmon of Ag@PVP, so that the Raman characteristic peak of gentamicin is shifted from 948 cm -1 to 786 cm -1 .

[0014] The distinguishing from other homologues refers to that under the detection system, the Raman characteristic peak of gentamicin appears at 786 cm -1 , and other homologues do not appear.

[0015] The other homologues include netilmicin, sisomicin, amikacin, streptomycin, tobramycin, neomycin and kanamycin.

[0016] With the increase of the concentration of the gentamicin sample solution, the SERS characteristic peak intensity of gentamicin is enhanced, a standard curve is established by using the relationship between the SERS characteristic peak intensity of gentamicin and the concentration, and the gentamicin solution is quantitatively analyzed and detected.

[0017] The linear detection range of the gentamicin is 10 -5 ~10 -9 M, the detection limit is 6.23 x 10 -10 M, and the relative standard deviation of the SERS intensity is less than 10%.

[0018] The pH of the SERS detection of the gentamicin sample solution mixed with the Ag@PVP core-shell nanoparticles and the Au@Ag NCs sol is 5-6.

[0019] The gentamicin sample solution is mixed with the Ag@PVP core-shell nanoparticles and the Au@Ag NCs sol for SERS detection within 10 s.

[0020] Compared with the prior art, the technical scheme of the present application has the following beneficial effects:

[0021] The present application uses Ag@PVP and Au@Ag NCs as the SERS substrate. The substrate not only has excellent SERS Raman enhancement effect, excellent stability and sensitivity, but also has the advantages of simple preparation process, low cost, high repeatability, excellent detection sensitivity and simple and easy-to-operate detection method. -5 ~10 -9 M, the detection limit is 6.23 x 10 -10 M, which is lower than the specified limit of gentamicin residue in animal food (200 μg / kg), and the relative standard deviation of the SERS intensity is less than 10%. DETAILED DESCRIPTION

[0022] Figure 1 Fig. 1A is a SEM image of Ag@PVP core-shell nanoparticles; Figure 1 Fig. 1B is a SEM image of Au@Ag NCs; Figure 1 Fig. 1C is a TEM image of Au@Ag NCs; Figure 1 Fig. 1D is a TEM image of Ag@PVP core-shell nanoparticles.

[0023] Figure 2 Fig. 2a is a SERS spectrum of Ag@PVP and 10 -5b) is the plasma resonance spectrum of the mixture of gentamicin and MgCl2; b) is the addition of Au@Ag NCs to Ag@PVP and 10 -5 Plasma resonance spectrum of M gentamicin mixed solution.

[0024] Figure 3 The SERS spectrum of the Ag@PVP-Gen-Au@Ag NCs system.

[0025] Figure 4 For Ag@PVP and 10 -5 SERS spectra of M gentamicin mixed in different volume ratios.

[0026] Figure 5 In the middle, A represents the SERS spectra of Ag@PVP-Gen-Au@Ag NCs system mixed at different volume ratios; Figure 5 Figure B shows the SERS spectra of gentamicin under different pH conditions.

[0027] Figure 6 In the middle A, the SERS spectra of gentamicin at different reaction times are shown. From top to bottom, a→f are (a) 5s, (b) 10s, (c) 40s, (d) 60s, (e) 80s, and (f) 100s. Figure 6 B represents the reaction time of gentamicin at 786 cm⁻¹ under different reaction conditions. -1 A line graph of the characteristic peak.

[0028] Figure 7 In the middle section, A represents the Ag@PVP-Gen-Au@Ag NCs system, and ten parallel measurements of gentamicin (10) were performed. -5 SERS spectrum of M); Figure 7 B represents ten parallel measurements of gentamicin (10) -5 M) at 786 cm -1 A bar chart showing the intensity of the SERS peak.

[0029] Figure 8 Figure A shows the SERS spectra of gentamicin at different concentrations, where the concentrations from a to e from top to bottom are (a) 10 -5 M (b) 10 -6 M (c) 10 -7 M (d) 10 -8 M (e) 10 -9 M; Figure 8 In the middle B, gentamicin was present at 786 cm. -1 A graph showing the relationship between SERS intensity and the negative logarithm of concentration at a given location.

[0030] Figure 9SERS spectra of different aminoglycoside antibiotic solutions mixed with Ag@PVP sol, from top to bottom are gentamicin, sisomicin, streptomycin, kanamycin, netilmicin, tobramycin, amikacin and neomycin.

[0031] Figure 10 SERS spectra of different aminoglycoside antibiotic solutions in the middle A, from top to bottom are gentamicin, netilmicin, sisomicin, amikacin, streptomycin, tobramycin, neomycin and kanamycin; Figure 10 In the middle B, the column chart of comparing gentamicin with other aminoglycoside antibiotics at Raman characteristic peak 786 cm -1 . DETAILED DESCRIPTION

[0032] In order to make the technical problems to be solved by the present application, technical solutions and beneficial effects more clear, explicit, the following will be combined with the drawings and examples, and the present application will be further described in detail.

[0033] The present application mixes gentamicin solutions with different concentrations with Ag@PVP and Au@Ag NCs in turn according to the best volume ratio, adjusts the mixed solution to the optimal pH value, excites with a suitable excitation wavelength, carries out surface enhanced Raman detection, and collects SERS spectra within the best reaction time. With the increase of the concentration of gentamicin, the intensity of the SERS characteristic peak will be enhanced, and a standard curve is established by using the relationship between the intensity of the SERS characteristic peak of gentamicin and the concentration, so as to carry out quantitative analysis and detection of the gentamicin solution. The Raman characteristic peak of gentamicin at 786 cm -1 is used for qualitative analysis and detection. The suitable excitation wavelength refers to the excitation wavelength of 785 nm; and the SERS characteristic peak of gentamicin at a specific wavelength is the SERS characteristic peak of gentamicin at 786 cm -1 .

[0034] The present application comprises the following steps:

[0035] 1) Preparation of Ag@PVP core-shell nanoparticles;

[0036] 2) Preparation of Au@Ag NCs sol;

[0037] 3) Mixing of gentamicin sample solution with Ag@PVP core-shell nanoparticles, and then adding Au@Ag NCs sol, distinguishing from other homologues by the Raman characteristic peak of gentamicin;

[0038] 4) Quantitative analysis and detection of gentamicin.

[0039] In step 1), the mass percentage concentration fraction of the sodium citrate is 0.8% to 1.5%, the volume ratio of sodium citrate to silver nitrate is 30 to 50, and the concentration of the silver nitrate solution is 1 to 5 mM; the reaction temperature is 90 to 100 °C, the reaction time is 50 to 60 min, and the stirring speed is 1000 to 1500 r / min; the Ag nanoparticles are spherical Ag nanoparticle sols with a particle size of about 50 nm. The mass concentration of the PVP solution is 1% to 4%, the volume of the PVP solution used is 40 to 50 μL, the stirring speed is 900 to 1200 r / min, and the reaction time is 20 to 30 min; the Ag@PVP is spherical Ag@PVP core-shell nanoparticles with a particle size of about 52.48 ± 10.34 nm.

[0040] In step 2), the concentration of the HAuCl4 solution used in the first step of the seed-mediated method for preparing Au@Ag NCs sol is 0.5 to 0.6 mM, the concentration of the cetyltrimethylammonium bromide (CTAB) solution is 200 to 250 mM, the concentration of the NaBH4 solution is 10 to 20 mM, the concentration of the CTAC solution is 200 to 250 mM, and the concentration of the ascorbic acid solution is 100 to 150 mM; the reaction temperature is 25 to 27 °C, and the stirring speed is 1000 to 1500 r / min. In the second step of the seed-mediated method for preparing Au@Ag NCs sol, the concentration of the CTAC solution is 20 to 25 mM, and the concentration of the silver nitrate solution is 2 to 5 mM; the concentration of the ascorbic acid solution in the ascorbic acid-CTAC solution is 50 to 60 mM, and the concentration of the CTAC solution is 40 to 50 mM; the reaction temperature is 60 to 70 °C, and the stirring speed is 1000 to 1500 r / min; the Au@Ag NCs are square Au@Ag NCs nanoparticles with a particle size of about 73.03 ± 10.26 nm.

[0041] The following gives specific examples.

[0042] 1) Synthesis of Ag@PVP core-shell nanoparticles

[0043] Silver sol was synthesized. 100 mL of a silver nitrate solution was taken in a single-necked round-bottom flask, heated to boiling reflux state under magnetic stirring with a heating mantle, and sodium citrate solution was rapidly added. The solution changed from colorless to milky white in about 1 min, and heating was continued for 50 to 60 min. After the milky white solution turned into a yellow-green solution, the heating mantle was removed to stop heating, and the round-bottom flask was naturally cooled to room temperature. The round-bottom flask was sealed with aluminum foil paper and stored in the dark.

[0044] Specifically, in the present embodiment, the mass percentage concentration fraction of sodium citrate is 0.8%, the volume ratio of sodium citrate to silver nitrate is 30, and the concentration of the silver nitrate solution is 1 mM.

[0045] Synthesis of Ag@PVP core-shell nanoparticles. Take the prepared silver sol 40 mL in a 100 mL round-bottom flask, add 40 μL of 1% PVP solution, and react at room temperature with a magnetic stirring speed of 1000 r / min for 20 min to obtain Ag@PVP core-shell nanoparticles with a shell thickness of about 3-5 nm. The particle size, shape, and shell thickness of the core-shell nanoparticles are characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and the nanoparticles are stored in the dark at 4°C.

[0046] 2) Synthesis of Au@Ag NCs sol

[0047] The specific method is divided into two steps. First, mix the HAuCl4 (5 mL, 0.5 mM) solution and the CTAB (5 mL, 200 mM) solution at room temperature and stir for 30 min, then add the frozen NaBH4 (0.6 mL, 10 mM) solution to the 10 mL aqueous solution containing HAuCl4 and CTAB to prepare a brown seed solution, then stir the seed solution in the dark at room temperature for 3 h (reaction temperature is 25-27°C, stirring speed is 1000-1500 r / min). Second, mix the HAuCl4 (20 mL, 0.5 mM) solution, the CTAC (20 mL, 200 mM) solution, and the ascorbic acid (15 mL, 100 mM) solution, stir the mixed solution in a water bath at room temperature (water bath temperature is 25-27°C) for 30 min to prepare a growth solution, then add the prepared seed solution to the above growth solution, mix gently for 30 s, then stir at room temperature for 15 min, and let the stirred solution stand for 30 min.

[0048] Second step, mix 3.5 mL of the above standing solution and 48.25 mL of the CTAC (20 mM) solution in a 100 mL glass bottle, heat in a 60°C water bath with magnetic stirring for 20 min, then simultaneously inject 50 mL of the AgNO3 (2 mM) solution and 50 mL of the ascorbic acid-CTAC solution (containing 50 mM ascorbic acid solution and 40 mM CTAC solution, each 25 mL) into the above solution at a rate of 0.2 mL / min, then react in a 60°C water bath for 4 h, and the reaction mixture changes from red to brown yellow. Finally, cool the glass bottle in an ice water bath, and store the synthesized Au@Ag NCs at 4°C.

[0049] Figure 1 Image A in the image is a SEM image of the prepared Ag@PVP core-shell nanoparticles. Figure 1 Image B in the image is a SEM image of the prepared Au@Ag NCs. Figure 1 C in the image is a TEM image of the prepared Au@Ag NCs. Figure 1 Image D is a TEM image of the prepared Ag@PVP core-shell nanoparticles. From... Figure 1 As can be seen in A and D, the Ag nanoparticles are surrounded by a relatively uniform PVP shell with a thickness of about 3 to 5 nm. The particle size of the Ag@PVP core-shell nanoparticles is about 52.48 ± 10.34 nm. Figure 1 Figures B and C show the final product of synthesized silver cubes, Au@Ag NCs. As can be seen from the figure, the size and uniformity of the particles are good, with a particle size of approximately 73.03 ± 10.26 nm.

[0050] 3) UV characterization of the Ag@PVP-Gen-Au@Ag NCs detection system

[0051] The Ag@PVP-Gen-Au@Ag NCs detection system was characterized by ultraviolet light. Figure 2 In the middle, 'a' represents Ag@PVP and 10. -5 b) is the plasma resonance spectrum of the mixture of gentamicin and MgCl2; b) is the addition of Au@Ag NCs to Ag@PVP and 10 -5 Plasma resonance spectrum of the M gentamicin mixed solution. (See image) Figure 2 As shown in Figure a, the absorption spectrum of Ag@PVP changed significantly after the addition of gentamicin, with a new plasmon resonance absorption peak appearing at ~772 nm. This phenomenon indicates that there is an electrostatic interaction between the positively charged amino groups in the gentamicin molecule and the negatively charged Ag@PVP nanoparticles. This interaction induces the nanoparticles to aggregate, forming aggregated nanostructures, which leads to the shift of the new surface plasmon resonance absorption peak to longer wavelengths, resulting in a significant redshift.

[0052] When in Ag@PVP and 10 -5 After adding Au@Ag NCs to the M gentamicin mixed solution, Figure 2 Multiple absorption peaks appeared in the plasmon resonance spectrum of Ag@PVP, located at 348 nm, 398 nm, 451 nm, 503 nm, and 735 nm. These peaks indicate that the interaction between Ag@PVP and gentamicin changed after the introduction of Au@AgNCs. Au@AgNCs modulated the surface plasmons of Ag@PVP, leading to the generation of new plasmon resonance modes. Compared to... Figure 2The peaks of the middle A changed obviously, especially the new absorption peaks at lower wavelengths (such as 348 nm and 398 nm), which may be due to the change of the local electronic environment of the detection system caused by the addition of Au@Ag NCs, resulting in the surface plasmon resonance absorption peak moving to the direction of shorter wavelength, and the obvious blue shift phenomenon appeared.

[0053] 4) Feasibility of Ag@PVP-Gen-Au@Ag NCs detection system

[0054] To verify the feasibility of the experiment, Ag@PVP, gentamicin (Gen) and Au@Ag NCs were mixed in turn and then Raman detection was carried out. From Figure 3 middle A, the Raman spectrum of Au@Ag NCs mixed with gentamicin is a straight line, which may be due to the fact that gentamicin does not interact with Au@Ag NCs, resulting in no characteristic peaks of gentamicin appearing in the Raman spectrum; when Ag@PVP and Gen are mixed, a Raman characteristic peak appears at 948 cm -1 , which may be because gentamicin induces the aggregation of Ag@PVP. In the process of aggregation, the Raman signal of C-H bending vibration is enhanced, so that gentamicin appears a Raman characteristic peak at 948 cm -1 ; When Au@Ag NCs are added to the mixture of Ag@PVP and Gen, the Raman characteristic peak is obviously enhanced at 786 cm -1 , which realizes the identification and differentiation of its homologues (see Figure 10 ), and can specifically detect gentamicin by SERS. This is because when gentamicin induces the aggregation of Ag@PVP, Au@Ag NCs regulate the surface plasmon of Ag@PVP, and the Raman signal of O-H stretching vibration is enhanced, which promotes the Raman characteristic peak of gentamicin to shift from 948 cm -1 to 786 cm -1 , which confirms the feasibility of the system; the Raman spectrum of Ag@PVP does not show SERS signal, indicating that the substrate has been successfully prepared.

[0055] By adjusting the addition order of Ag@PVP and Au@Ag NCs, it is further verified that the shift of the Raman characteristic peak of gentamicin is due to the fact that Au@Ag NCs regulate the surface plasmon of Ag@PVP after gentamicin induces the aggregation of Ag@PVP. From the comparison of Figure 3 middle B, it can be seen that when Ag@PVP and Au@Ag NCs are mixed first and then gentamicin is added, the Raman characteristic peak of gentamicin is at 948 cm -1The Raman characteristic peaks were significantly weaker, indicating that the order of addition of Ag@PVP and Au@Ag NCs has a certain impact on the detection of gentamicin.

[0056] 5) Optimization of the optimal volume ratio

[0057] The SERS substrate (Ag@PVP, Au@Ag NCs) represents the concentration of the stock synthesis solution, and the concentration of the gentamicin solution is 10. -5 M. First, Ag@PVP and gentamicin solution were mixed at different volume ratios (1:2, 1:1.5, 1:1, 1:0.5) to determine the optimal volume ratio of Ag@PVP to gentamicin. See Figure 4 It can be seen that when the volume ratio of Ag@PVP to gentamicin solution is 1:1, gentamicin induces Ag@PVP to aggregate at 948 cm⁻¹. -1 A strong Raman characteristic peak appeared at that location.

[0058] Subsequently, Au@Ag NCs sol was added to the Ag@PVP and gentamicin mixture, and the volume ratios of Au@Ag NCs to the Ag@PVP and gentamicin mixture were adjusted to 2:1, 1.5:1, 1:1, 0.67:1, and 0.5:1, respectively. SERS spectra were then acquired. See [link / reference]. Figure 5 As shown in Figure A, when the volume ratio of Au@AgNCs to the mixed solution of Ag@PVP and gentamicin reaches 1:1, Au@AgNCs modulates the surface plasmon resonance of Ag@PVP, causing the Raman characteristic peak of gentamicin to rise from 948 cm⁻¹. -1 Displaced to 786 cm -1 This allows them to be distinguished from their homologues. Therefore, in subsequent experiments, the volume ratio of Au@Ag NCs to the Ag@PVP and gentamicin mixture was kept constant at 1:1.

[0059] 6) Optimization of optimal pH

[0060] The volume ratio of Au@Ag NCs to Ag@PVP and gentamicin was kept constant at 1:1. The pH of the mixture was adjusted to 2, 4, 5, 6, 8 and 10. The surface-enhanced Raman spectrum of gentamicin was scanned to determine the optimal pH.

[0061] Figure 5 Figure B shows the SERS spectra of gentamicin measured under different pH conditions. It can be seen from the figure that the intensity of the surface-enhanced Raman signal peak of gentamicin is strongest at pH=5, and weakest at pH values ​​of 6, 8, or 10. Figure 5 (In pH B), the signal intensity of the Raman characteristic peaks decreased significantly. Therefore, the optimal pH for the gentamicin detection system was fixed at 5.

[0062] 7) Optimization of reaction time

[0063] The volume ratio of fixed Au@Ag NCs and Ag@PVP and gentamicin mixed solution was 1:1, the pH value of the mixed solution was 5, and the reaction time was adjusted to 5 s, 10 s, 40 s, 60 s, 80 s, and 100 s, respectively. The SERS spectrum of gentamicin was scanned to determine the optimal reaction time.

[0064] Figure 6 In the middle A, the SERS spectra of gentamicin under different reaction time conditions were measured, from top to bottom a→f, (a) 5 s, (b) 10 s, (c) 40 s, (d) 60 s, (e) 80 s, and (f) 100 s. Combined with Figure 6 In the middle B, it can be seen that the SERS detection effect of gentamicin is best when the reaction time is 0-10 s. Therefore, the optimal reaction time of the gentamicin detection system is within 10 s.

[0065] 8) SERS substrate uniformity detection

[0066] The volume ratio of fixed Au@Ag NCs and Ag@PVP and gentamicin mixed solution was 1:1, the pH value was adjusted to 5, and the SERS spectrum was collected randomly ten times on the substrate with an excitation wavelength of 785 nm. The SERS peak intensity of gentamicin collected ten times in parallel was compared to determine the uniformity of the SERS substrate.

[0067] Figure 7 In the middle A, the SERS spectra of gentamicin were measured ten times in parallel, Figure 7 In the middle B, the SERS peak intensity of gentamicin at 786 cm -1 was measured ten times in parallel. It can be seen that the relative standard deviation (RSD) calculated from the SERS characteristic peak intensity of the 10 parallel samples is 3.29% at 786 cm -1 , which is less than 10%, indicating that the uniformity of the SERS substrate is good and can be applied to the quantitative analysis of the surface enhanced Raman of gentamicin.

[0068] 9) SERS detection of gentamicin

[0069] The volume ratio of fixed Au@Ag NCs and Ag@PVP and gentamicin mixed solution was 1:1, the pH value was adjusted to 5, and the SERS spectrum was collected under excitation with an excitation light wavelength of 785 nm. The SERS spectra of gentamicin with different concentrations were measured.

[0070] Figure 8 In the middle A, the SERS spectra of gentamicin with different concentrations were measured, where the concentrations from top to bottom a→e were 10 -5 M, 10-6 M, 10 -7 M, 10 -8 M, 10 -9 M, it can be seen that the surface-enhanced Raman peak intensity of gentamicin is stronger with the increase of concentration.

[0071] Figure 8 B is the standard curve established after detecting different concentration standard solutions of gentamicin, and the standard curve is linear in the range of 10 -5 ~ 10 -9 M, the intensity of the characteristic peak of gentamicin 786 cm -1 is negatively correlated with the concentration of gentamicin, showing a positive correlation (y = 1349.48 x + 13468.23), R 2 = 0.9968. In addition, the detection limit (LOD) is 6.23 x 10 -10 M.

[0072] 10) Selectivity of SERS substrate (Ag@PVP, Au@Ag NCs) to the same class of aminoglycoside antibiotics

[0073] Take Ag@PVP sol and mix it with 10 -5 M gentamicin, netilmicin, sisomicin, amikacin, streptomycin, tobramycin, neomycin and kanamycin, and fix the volume ratio at 1:1. Adjust the pH value to 5-6. Get the surface-enhanced Raman spectra of gentamicin, netilmicin, sisomicin, amikacin, streptomycin, tobramycin, neomycin and kanamycin at the excitation wavelength of 785 nm.

[0074] From Figure 9 it can be seen that when Ag@PVP sol is mixed with different aminoglycoside antibiotics, it can be observed from the Raman spectrum that Ag@PVP sol as SERS substrate cannot distinguish the same class of aminoglycoside antibiotics.

[0075] Take Ag@PVP sol and mix it with 10 -5 M gentamicin, netilmicin, sisomicin, amikacin, streptomycin, tobramycin, neomycin and kanamycin, and then add Au@Ag NCs sol. Fix the volume ratio of Au@Ag NCs to Ag@PVP and antibiotic mixed solution at 1:1. Adjust the pH value to 5-6. Get the surface-enhanced Raman spectra of gentamicin, netilmicin, sisomicin, amikacin, streptomycin, tobramycin, neomycin and kanamycin at the excitation wavelength of 785 nm.

[0076] Figure 10SERS spectra of gentamicin, netilmicin, sisomicin, amikacin, streptomycin, tobramycin, neomycin and kanamycin solutions, wherein A is different; Figure 10 SERS spectra of gentamicin and other aminoglycoside antibiotics, wherein B is the Raman characteristic peak 786 cm -1 of gentamicin and other aminoglycoside antibiotics detected under the same conditions. Figure 10 It can be seen that under acidic conditions, the Au@Ag NCs are added to the mixed solution of Ag@PVP sol and sample solution, only gentamicin shows obvious Raman characteristic peaks at 786 cm -1 , while other aminoglycoside antibiotics do not have such characteristics. Among them, amikacin, streptomycin, tobramycin, neomycin and kanamycin have smaller characteristic peaks on the left side of 786 cm -1 , and the characteristic peaks of netilmicin and sisomicin appear at 1697 cm -1 , so under acidic conditions (pH=5-6), Ag@PVP and Au@Ag NCs can be used as SERS substrates to distinguish gentamicin, netilmicin, sisomicin, amikacin, streptomycin, tobramycin, neomycin and kanamycin, and it is proved that the detection system has specificity for gentamicin.

[0077] The present application uses Ag@PVP and Au@Ag NCs as SERS substrates to realize qualitative and quantitative analysis of gentamicin. The linear detection range of gentamicin is 10 -5 -10 -9 M, the detection limit is 6.23 × 10 -10 M, and the relative standard deviation of SERS intensity is less than 10%. Ag@PVP and Au@Ag NCs as SERS substrates have the advantages of low cost, good stability, high detection efficiency, etc., and can be used for practical detection and popular application in SERS detection of antibiotics and the like. Moreover, the method is simple to operate, accurate and sensitive, does not require complex operation technology, and can meet the requirements of large-scale and rapid analysis and detection.

Claims

1. A method for surface enhanced Raman spectroscopy (SERS) detection of gentamicin based on multi-dimensional plasmonic coupling system of Ag@PVP and Au@Ag NCs, characterized in that, The method comprises the following steps: 1) preparing Ag@PVP core-shell nanoparticles; 2) preparing Au@Ag NCs sol; 3) mixing the gentamicin sample solution with the Ag@PVP core-shell nanoparticles, then adding the Au@Ag NCs sol, and distinguishing the gentamicin from other homologues through the Raman characteristic peaks of the gentamicin; 4) quantitative analysis and detection of the gentamicin; Wherein, gentamicin sample solution mixed with Ag@PVP core-shell nanoparticles, gentamicin induced Ag@PVP agglomeration, gentamicin in 948 cm -1 Raman characteristic peak appears; After adding Au@Ag NCs sol, Au@Ag NCs modulate the surface plasmon of Ag@PVP, which makes the Raman characteristic peaks of gentamicin shift from 948 cm -1 to 786 cm -1 ; The distinguishing from other homologues means that the Raman characteristic peak position of gentamicin appears at 786 cm -1 under the detection system, and other homologues do not appear.

2. The method of SERS detection of gentamicin using the multi-dimensional plasmonic coupling system based on Ag@PVP and Au@Ag NCs as claimed in claim 1, characterized in that: In step 1), the silver sol is prepared by using the sodium citrate reduction method, then the PVP is added into the silver sol, and the Ag@PVP core-shell nanoparticles are prepared.

3. The method of SERS detection of gentamicin using Ag@PVP and Au@Ag NCs based multi-dimensional plasmonic coupling system as claimed in claim 1, wherein: In step 2), the gold seed solution and the growth solution are respectively synthesized, the gold growth solution is obtained after the two solutions are mixed and placed, then the gold growth solution is reacted with the CTAC solution, and the Au@Ag NCs sol is prepared by simultaneously injecting the silver nitrate solution and the ascorbic acid-CTAC solution.

4. The method of SERS detection of gentamicin using Ag@PVP and Au@Ag NCs based multi-dimensional plasmonic coupling system as claimed in claim 1, wherein: The other homologues include netilmicin, sisomicin, amikacin, streptomycin, tobramycin, neomycin and kanamycin.

5. The method of SERS detection of gentamicin using Ag@PVP and Au@Ag NCs based multi-dimensional plasmonic coupling system as claimed in claim 1, wherein: With the increase of the concentration of the gentamicin sample solution, the SERS characteristic peak intensity of the gentamicin is enhanced, the standard curve is established by using the relationship between the SERS characteristic peak intensity and the concentration of the gentamicin, and the quantitative analysis and detection of the gentamicin solution are carried out.

6. The method of SERS detection of gentamicin using Ag@PVP and Au@Ag NCs based multi-dimensional plasmonic coupling system as claimed in claim 1, wherein: The linear detection range of gentamicin is 10 -5 ~ 10 -9 M, the detection limit is 6.23 x 10 -10 M, the relative standard deviation of SERS intensity is less than 10%.

7. The method of SERS detection of gentamicin using Ag@PVP and Au@Ag NCs based multi-dimensional plasmonic coupling system as claimed in claim 1, wherein the method comprises of the following steps: a. Synthesis of Ag@PVP and Au@Ag NCs; b. Preparation of the sample solution; c. Preparation of the SERS substrate; d. SERS measurement; and e. Data analysis. The pH of the SERS detection after the gentamicin sample solution is mixed with the Ag@PVP core-shell nanoparticles and the Au@Ag NCs sol is 5-6.

8. The method of SERS detection of gentamicin using Ag@PVP and Au@Ag NCs based multi-dimensional plasmonic coupling system as claimed in claim 1, wherein: The SERS detection of the gentamicin sample solution mixed with the Ag@PVP core-shell nanoparticles and the Au@Ag NCs sol is carried out within 10 s.

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