Preparation of composite sandwich nano film Au / Ag / Ag and application of composite sandwich nano film Au / Ag / Ag in SERS (Surface Enhanced Raman Scattering) detection of netilmicin

Abstract

The invention discloses preparation of a composite sandwich nano film Au / Ag / Ag and application of the composite sandwich nano film Au / Ag / Ag in SERS (Surface Enhanced Raman Scattering) detection of netilmicin, and relates to the field of analysis and detection.The preparation method comprises the steps that firstly, a gold film is evaporated on one layer of polyvinylidene fluoride porous film (CVDF) in a vacuum mode, silver films are synthesized on the two faces of the other layer of CVD film through a glucose reduction method, and the two layers of CVD polymer films with the long gold and silver films are covered and stacked; a composite polymer sandwich nano film Au / Ag / Ag is constructed, surface plasmas of the film are promoted to be mutually coupled by regulating and controlling the thickness of the film, and the SERS detection sensitivity can be greatly improved. Under optimal experimental conditions, the linear detection range of netilmicin is 10 <-5 >-10 <-9 > M, the detection limit is 4.35 * 10 <-10 > M, and the relative standard deviation of SERS intensity is less than 10%. The method has the advantages of high sensitivity, high stability, high repeatability and good enhancement effect, and meets the requirements of large-scale and rapid analysis and detection.

Description

Technical Field

[0001] The invention relates to the field of analysis and detection, and in particular to the preparation of a composite sandwich nanofilm Au / Ag / Ag and the application of the composite sandwich nanofilm in SERS detection of netilmicin. Background Art

[0002] Netilmicin (NET) is a semi-synthetic aminoglycoside antibiotic. It is currently widely used in the clinical treatment of respiratory and digestive tract diseases caused by bacteria such as Escherichia coli and Proteus. It is also used for urinary tract infections and secondary infections caused by multisystemic wasting syndrome after weaning in various farm animals. It is an antibiotic widely used in health care and agriculture, but its accumulation in the environment poses a threat to human health. However, due to its stable chemical structure and water solubility, netilmicin is difficult to be naturally degraded or completely removed. Long-term residues in the body can induce a variety of toxic reactions in the human body, including ototoxicity, nephrotoxicity, neurotoxicity, etc. If it is used irrationally, it is easy to cause netilmicin to accumulate in the environment, thereby endangering human health. Therefore, it is necessary to develop a reliable, highly sensitive, and portable detection method to analyze and quantify netilmicin residues in environmental samples.

[0003] At present, the main detection methods for netilmicin are: capillary electrophoresis, high performance liquid chromatography-pulsed electrochemical detection and mass spectrometry. Capillary electrophoresis has the advantages of high resolution and small sample usage, but aminoglycoside antibiotics have similar structures and charge characteristics, their separation in capillary electrophoresis may be limited, and the equipment cost is high. High performance liquid chromatography-pulsed electrochemical detection can sensitively detect aminoglycoside antibiotics without derivatization, but due to the instability of the analytical signal, the detection has certain limitations, and the selectivity is slightly poor, the equipment cost is high, and the separation speed is slow. Mass spectrometry equipment is expensive, the operation is complicated, and the sample pretreatment requirements are high. These analytical instrument-based methods have high sensitivity and specificity, but their poor portability makes them unsuitable for on-site detection. In addition, the detection process also requires cumbersome pretreatment steps and professional operators, which also limits the large-scale application of on-site monitoring. Therefore, it is urgent to establish a reliable, highly sensitive and portable detection method to accurately identify netilmicin in environmental and food samples. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems in the prior art and provide a surface enhanced Raman scattering (SERS) detection method for detecting netilmicin, using a composite sandwich nanofilm Au / Ag / Ag as a SERS substrate to perform quantitative analysis of netilmicin. The linear detection range of netilmicin is 10 -5 ~10 -9 M, detection limit is 4.35×10 -10M, the relative standard deviation of SERS intensity is less than 10%. The composite sandwich nanofilm Au / Ag / Ag as a SERS substrate has the advantages of simplicity, convenience, controllability, and high detection efficiency. In addition, this method is easy to operate, has high accuracy and sensitivity, does not require complex operating techniques, and can meet the requirements of large-scale and rapid analysis and detection.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] The preparation of the composite sandwich nanofilm Au / Ag / Ag comprises the following steps:

[0007] 1) Prepare an Au film on top of the first CVDF film;

[0008] 2) Synthesize Ag film on the front and back of the second CVDF film;

[0009] 3) Compounding the two films, with the Au film on top and the Ag film on the bottom, to form a composite sandwich nanofilm Au / Ag / Ag.

[0010] In step 1), the thickness of the first CVDF film is 100 to 800 μm, and the thickness of the Au film is 20 to 180 nm.

[0011] In step 2), the Ag film is prepared by glucose reduction method. First, ammonia water is added to the AgNO3 solution, and then the CVDF membrane is added and left to stand for activation and then placed in glucose solution for reduction reaction.

[0012] The concentration of the ammonia water is 0.1-0.6M.

[0013] The composite sandwich nanofilm Au / Ag / Ag is used in SERS detection of netilmicin.

[0014] The invention uses composite sandwich nanofilm Au / Ag / Ag as SERS substrate, drips AgNPs sol and netilmicin solution on the upper film, mixes them, drips NaCl solution, and performs SERS detection at the optimal excitation wavelength.

[0015] As the concentration of the sample solution increases, the Raman peak of the sample to be tested at a specific wavelength gradually increases. A standard curve is established using the relationship between the intensity of the Raman characteristic peak of the sample to be tested and the amount of the sample to be tested. The sample to be tested is then quantitatively analyzed and tested, and netilmicin is distinguished from similar aminoglycoside antibiotics based on the different Raman characteristic peaks.

[0016] The optimal excitation wavelength refers to the 785 nm excitation wavelength; the Raman peak of netilmicin at a specific wavelength refers to the Raman peak of netilmicin at 1697 cm -1 The characteristic Raman peak at .

[0017] The linear detection range of netilmicin of the present invention is 10 -5 ~10 -9 M, detection limit is 4.35×10 -10 M, the relative standard deviation of SERS intensity is less than 10%.

[0018] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0019] The present invention prepares a composite sandwich nanofilm Au / Ag / Ag as a SERS substrate, captures the SERS signal of netilmicin by using AgNPs, adjusts the density of silver grown on a CVDF-Ag film prepared by a glucose reduction method, the thickness of the CVDF film, and the thickness of a CVDF-Au film prepared by a vacuum evaporation method, and changes the distance between the CVDF-Ag film and the CVDF-Au film to promote magnetic field superposition, thereby enhancing the SERS signal of netilmicin and reducing the detection limit. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 In the figure, the thickness of CVDF membranes from A to E are 100 μm, 200 μm, 400 μm, 600 μm, and 800 μm respectively;

[0021] Figure 2 A in the middle is the SEM image of the CVDF membrane; Figure 2 B is the SEM image of CVDF-Ag film; Figure 2 C in the middle is the SEM image of CVDF-Au; Figure 2 D in the middle is the SEM image of AgNPs sol; Figure 2 E to J in the middle are SEM images of CVDF-Ag films with different particle sizes of 40nm, 20nm, 30nm, 50nm, 60nm, and 100nm, respectively;

[0022] Figure 3 A in the middle is the SERS spectra of AgNPs sol, Ag film and Au film and their detection of netilmicin. Figure 3 Middle B is the SERS spectra of different combinations of composite sandwich nanofilm Au / Ag / Ag;

[0023] Figure 4 The SERS spectra of the composite sandwich nanofilm Au / Ag / Ag separated by netilmicin under different thickness CVDF conditions;

[0024] Figure 5 The SERS spectra of netilmicin under Au conditions with different thicknesses of composite sandwich nanofilm Au / Ag / Ag;

[0025] Figure 6 The SERS spectra of the composite sandwich nanofilm Au / Ag / Ag detected by Netilmicin under different ammonia concentrations;

[0026] Figure 7 The SERS spectra of the composite sandwich nanofilm Au / Ag / Ag detection of netilmicin under different NaCl addition amounts;

[0027] Figure 8 A in the middle is the SERS spectrum of netilmicin measured ten times in parallel on the composite sandwich nanofilm Au / Ag / Ag; Figure 8 B is a parallel measurement of netilmicin ten times (10 -5 M) at 1697 cm -1 Histogram and relative standard deviation of SERS peak intensity at ;

[0028] Fig. 9 A in the middle is the SERS spectra of different concentrations of netilmicin on the composite sandwich nanofilm Au / Ag / Ag. Fig. 9 B is netilmicin at 1697 cm -1 The linear relationship between the SERS intensity at and the negative logarithm of the concentration;

[0029] Fig.10 SERS spectra of different aminoglycoside antibiotic solutions in the composite sandwich nanomembrane Au / Ag / Ag. DETAILED DESCRIPTION

[0030] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0031] The present invention comprises the following steps:

[0032] 1) Preparation of AgNPs sol by sodium citrate reduction method;

[0033] 2) Preparation of nanocellulose fine-tuned polyvinylidene fluoride porous membrane (CVDF membrane);

[0034] 3) Prepare an Au film on a layer of CVDF;

[0035] 4) Another layer of CVDF film is synthesized with Ag film on the front and back;

[0036] 5) Compounding the two layers of membranes to form a composite sandwich nanomembrane Au / Ag / Ag, which is used as a substrate for SERS detection;

[0037] 6) SERS quantitative analysis and detection on composite sandwich nanofilm Au / Ag / Ag.

[0038] In step 1), a sodium citrate reduction method is used to control the reaction parameters to obtain AgNPs sol with uniform particle size. The specific steps are: take an AgNO3 solution in a flask, stir and heat to boiling reflux state, add sodium citrate until the solution becomes a slightly green opaque liquid, continue to heat and reflux, stop heating, naturally cool to room temperature, and seal and store in a dark place.

[0039] In step 2), the CVDF membrane is prepared by a phase inversion method.

[0040] In step 3), the CVDF-Au film is prepared by vacuum evaporation.

[0041] In step 4), the silver film is prepared by glucose reduction method, ammonia water is added to the AgNO3 solution, and then the CVDF membrane is added and placed in glucose solution for reduction reaction after being activated.

[0042] In step 6), under the optimal experimental conditions, a standard curve is established with the logarithmic values ​​of the series concentrations of the sample solution as the abscissa and the peak intensity of the sample solution at the strongest characteristic peak as the ordinate, and the sample solution to be tested is quantitatively analyzed and detected.

[0043] Specific examples are given below.

[0044] Example 1

[0045] 1) Preparation of AgNPs sol by sodium citrate reduction method;

[0046] Ag nanoparticle sol with uniform particle size can be obtained by controlling the concentration of silver nitrate to 1-3 mM, the reaction temperature to 90-100 °C, the reaction time to 30-80 min, and the stirring speed to 1000-1500 r / min. The specific steps are: take AgNO3 solution in a flask, stir and heat to boiling reflux state, add sodium citrate until the solution becomes a slightly green opaque liquid, continue heating and reflux for 50-60 min, stop heating, cool naturally to room temperature, and seal and store in a dark place.

[0047] Specifically, in this embodiment, the mass concentration of sodium citrate is 1%, the concentration of silver nitrate is 1 mM, the volume ratio of sodium citrate to silver nitrate is 1:30; the reaction temperature is 100°C, and the reaction time is 80 min.

[0048] 2) Preparation of CVDF membrane;

[0049] Using dimethylformamide (DMF) as solvent, polyvinyl pyrrolidone (PVP), polyvinylidene fluoride (PVDF) and cellulose nanocrystals (CNC) were strongly dissolved in DMF for at least 10 to 12 hours. The prepared membrane solution was poured into a culture dish and left to stand at room temperature for 10 to 12 hours to remove bubbles. After being exposed to air for 10 seconds, the culture dish was immersed in a deionized water bath, polymer precipitation occurred to form a membrane, and the membrane was washed and stored in deionized water. By controlling the amount of membrane solution used, the membrane thickness was 100 to 800 μm. The mass of PVP was 0.5 to 1 g, the mass of PVDF was 2 to 10 g, the mass of DMF was 10 to 60 g, and the mass of CNC was 0.01 to 0.05 g.

[0050] Specifically, in this example, 0.5 g PVP, 7.5 g PVDF and 0.03 g CNC were strongly dissolved in 42 g DMF.

[0051] 3) Preparing an Au film on a layer of CVDF film;

[0052] After freeze drying the CVDF membrane for 20 to 24 h, place it in an electron beam vacuum evaporation instrument at a vacuum degree of 4.5 × 10 -7 ~5.0×10 -7 Torr, and vacuum evaporation was carried out at an electron beam power of 26% to 28%.

[0053] 4) another layer of CVDF film is synthesized with Ag film on the front and back;

[0054] The ammonia concentration of the silver ammonia solution can be controlled to be 0.1~0.6M, the volume ratio of the silver ammonia solution to glucose to be 1:30~50, the reaction temperature to be 20~30℃, the reaction time to be 12~30h, and the oscillation speed to be 100~150 r / min. Specifically, a 100~300mM AgNO3 solution is prepared in a beaker, and ammonia water is added dropwise until it becomes clear and transparent. Then, a CVDF membrane is added and allowed to stand for activation for 30min, and then placed in a glucose solution for reduction reaction, and then placed in a shaker for reaction, and finally stored in a cool place.

[0055] More specifically, in this embodiment, the mass concentration fraction of the ammonia water is 0.5M, the volume ratio of the silver ammonia solution to glucose is 1:30; the concentration of the silver nitrate is 100mM; the reaction temperature can be 22°C, the reaction time is 15 h, and the oscillation speed is 150 r / min.

[0056] 5) Compounding the two layers of membranes to form a composite sandwich nanomembrane Au / Ag / Ag, which is used as a substrate for SERS detection;

[0057] The Au film was prepared on the top of a CVDF film and the Ag film was synthesized on the front and back of another CVDF film. The Au film was on the top and the Ag film was on the bottom to form a composite sandwich nanofilm Au / Ag / Ag as a substrate for SERS detection. AgNPs sol and netilmicin solution were added to the upper film and then 1M NaCl solution was added to adjust the mixture. SERS detection was performed at the optimal excitation wavelength.

[0058] Figure 1 As characterized by a high-precision vernier caliper, the thickness of the CVDF membrane prepared from A→E in the figure is 100μm, 200μm, 400μm, 600μm, and 800μm. CVDF membranes of different thicknesses were successfully produced.

[0059] Figure 2 A in the middle is the SEM image of the CVDF membrane. It can be seen that the pore size of the CVDF membrane is uniform and rough, the state of the CVDF membrane is smooth and stretched, and the CVDF membrane is successfully manufactured. Figure 2 B is the SEM image of the CVDF-Ag film, which shows that dense and uniform silver nanoparticles are successfully grown on the surface of the CVDF film to form a silver film; Figure 2 C in the middle is the SEM image of CVDF-Au, which shows that the gold film on the CVDF membrane is evenly prepared and successfully evaporated on the CVDF membrane; Figure 2 D in the middle is the SEM image of the AgNPs sol, which shows that the AgNPs sol is uniformly prepared and successfully reduced to Ag nanoparticles, and the particle size of the Ag nanoparticles is about 50±10nm; Figure 2 E to J in the middle are SEM images of CVDF-Ag films with different particle sizes of 40nm, 20nm, 30nm, 50nm, 60nm, and 100nm respectively. The particle size of the silver nanoparticles on the CVDF-Ag film is controlled by adjusting different ammonia concentrations. It can be seen that when the silver nanoparticles grown on the CVDF film are 60nm, the silver nanoparticles grow most uniformly and densely.

[0060] Figure 3 A in the middle is the SERS spectra of AgNPs sol, Ag film and Au film and their detection of netilmicin. It can be seen that the SERS signal of the Ag film is the best. Figure 3 Middle B shows the SERS spectra of different combinations of composite sandwich nanofilms Au / Ag / Ag. It can be seen that the SERS enhancement effect is best when the Au film is on top and the Ag film is on the bottom.

[0061] Example 2

[0062] SERS detection of netilmicin in composite sandwich nanofilm Au / Ag / Ag under different CVDF thickness conditions.

[0063] The concentration of the netilmicin solution was fixed at 10 -5M, adjust the thickness of the CVDF film prepared with Au film, scan the SERS spectrum of netilmicin on the composite sandwich nanofilm Au / Ag / Ag combined SERS substrate, and determine the optimal thickness of the CVDF film.

[0064] The concentration of the netilmicin solution was fixed at 10 -5 M, adjust the thickness of the CVDF film to 100 μm to 800 μm, collect SERS spectra, and Figure 4 It can be seen that when the thickness of the CVDF film is 200 μm, the SERS intensity of netilmicin reaches the highest. Therefore, in the subsequent examples, a composite sandwich nanofilm Au / Ag / Ag film combined SERS substrate separated by a CVDF film with a thickness of 200 μm is used to detect netilmicin.

[0065] Example 3

[0066] SERS detection of netilmicin under Au conditions with different thicknesses of composite sandwich nanofilm Au / Ag / Ag.

[0067] The concentration of the netilmicin solution was fixed at 10 -5 M, and the thickness of the CVDF film was fixed at 200 μm, the thickness of the Au film vacuum evaporated on the surface of the CVDF membrane was adjusted to 20nm, 60nm, 100nm, 140nm, and 180nm, and the SERS spectrum of netilmicin on the composite sandwich nanomembrane Au / Ag / Ag combined SERS substrate was scanned to determine the optimal thickness of the Au film.

[0068] Figure 5 Figure 2 is the SERS spectra of netilmicin under different Au thickness conditions of composite sandwich nanofilm Au / Ag / Ag. It can be seen that when the thickness of the Au film is 60nm, the SERS intensity of netilmicin is the strongest. Therefore, the optimal Au film thickness of the netilmicin detection system is 60nm.

[0069] Example 4

[0070] SERS spectra of netilmicin detected by Au / Ag / Ag composite sandwich nanomembrane under different ammonia concentrations.

[0071] The concentration of the netilmicin solution was fixed at 10 -5 M, fixed the thickness of the CVDF film at 200 μm, fixed the thickness of the vacuum evaporated Au film on the surface of the CVDF film at 60 nm, adjusted the concentration of ammonia water during the preparation of the CVDF-Ag film to 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, and 0.6 M, scanned the SERS spectrum of netilmicin on the composite sandwich nanomembrane Au / Ag / Ag film combined SERS substrate, and determined the concentration of ammonia water during the preparation of the CVDF-Ag film.

[0072] Figure 6 This is the SERS spectrum of the composite sandwich nanomembrane Au / Ag / Ag detection of Netilmicin under different ammonia concentration conditions. It can be seen that when the concentration of ammonia is 0.5 M, the SERS intensity of Netilmicin is the strongest. Therefore, the optimal ammonia concentration of the detection system of Netilmicin is 0.5 M.

[0073] Example 5

[0074] SERS spectra of netilmicin detected by composite sandwich nanomembrane Au / Ag / Ag at different NaCl addition amounts.

[0075] The concentration of the netilmicin solution was fixed at 10 -5 M, the thickness of the CVDF film is 200 μm, the thickness of the vacuum evaporated Au film on the surface of the CVDF film is 60 nm, the concentration of ammonia water during the preparation of the CVDF-Ag film is 0.5 M, the addition amount of 1M NaCl solution is adjusted to 0.5 μL, 1 μL, 2 μL, 3 μL, and 4 μL, and the SERS spectrum of netilmicin on the composite sandwich nanomembrane Au / Ag / Ag film combined SERS substrate is scanned to determine the amount of NaCl added.

[0076] Figure 7 This is the SERS spectrum of the composite sandwich nanofilm Au / Ag / Ag detection of Netilmicin at different NaCl addition amounts. It can be seen that when the NaCl addition amount is 1 μL, the SERS intensity of Netilmicin is the strongest, so the optimal NaCl addition amount for the Netilmicin detection system is 1 μL.

[0077] Example 6

[0078] The uniformity of the composite sandwich nanomembrane Au / Ag / Ag substrate.

[0079] The concentration of the netilmicin solution was fixed at 10 -5 M, the thickness of the CVDF film is 200 μm, the thickness of the vacuum evaporated Au film on the surface of the CVDF film is 60 nm, the concentration of ammonia water during the preparation of the CVDF-Ag film is 0.5 M, the amount of 1M NaCl solution added is adjusted to 1 μL, the SERS spectrum of netilmicin on the composite sandwich nanomembrane Au / Ag / Ag film combined SERS substrate is scanned, and the SERS spectrum is randomly collected ten times on the substrate with an excitation wavelength of 785 nm. The SERS peak intensities of netilmicin collected ten times in parallel are compared to determine the uniformity of the SERS substrate.

[0080] Figure 8 It can be seen that the peak shape of netilmicin measured ten times is consistent and has good stability.

[0081] Figure 8 B in the figure shows ten parallel measurements of netilmicin at 1697 cm -1From the bar graph of SERS peak intensity, it can be seen that the relative standard deviation (RSD) calculated from the characteristic SERS peak intensity of 10 groups of parallel samples is 4.12%, which is less than 10%, indicating that the uniformity of the SERS substrate is good and can be applied to the SERS quantitative analysis of netilmicin.

[0082] Example 7

[0083] Composite sandwich nanomembrane Au / Ag / Ag for detection of netilmicin.

[0084] The concentration of the netilmicin solution was fixed at 10 -5 M, the thickness of the CVDF film is 200 μm, the thickness of the vacuum evaporated Au film on the surface of the CVDF film is 60 nm, the concentration of ammonia water during the preparation of the CVDF-Ag film is 0.5 M, the amount of 1 M NaCl solution added is adjusted to 1 μL, and the SERS spectra are collected on the substrate with an excitation wavelength of 785 nm. The SERS spectra of netilmicin at different concentrations are measured.

[0085] Fig. 9 It can be seen that as the concentration decreases, the SERS intensity of netilmicin decreases.

[0086] Fig. 9 B is the standard curve established after testing different concentrations of standard solutions of netilmicin. -5 ~10 -9 M range, netilmicin 1697 cm -1 The intensity of the characteristic peak has a negative logarithmic relationship with the concentration of netilmicin, showing a positive correlation (y = 2877.56X + 27229.94), R 2 = 0.9966. In addition, the detection limit (LOD) is 4.35×10 -10 M.

[0087] Example 8

[0088] Selectivity of composite sandwich nanomembrane Au / Ag / Ag for netilmicin-like and different antibiotic solutions.

[0089] The concentration of netilmicin, gentamicin, streptomycin, tobramycin, and neomycin solution was fixed at 10 -5 M, the thickness of the CVDF film is 200 μm, the thickness of the vacuum evaporated Au film on the surface of the CVDF film is 60 nm, the concentration of ammonia water during the preparation of the CVDF-Ag film is 0.5 M, the amount of 1 M NaCl solution added is adjusted to 1 μL, and the SERS spectra of different antibiotics are collected on the substrate with an excitation wavelength of 785 nm.

[0090] Fig.10Figure 2 is the SERS spectra of netilmicin, gentamicin, streptomycin, tobramycin, and neomycin solutions. As can be seen from the figure, compared with the characteristic peaks of gentamicin, streptomycin, tobramycin, and neomycin, netilmicin has a unique peak at 1697 cm -1 Characteristic peaks can be detected in the sample, so netilmicin can be distinguished from gentamicin, streptomycin, tobramycin and neomycin.

Claims

1. Preparation of composite sandwich nanofilm Au / Ag / Ag, characterized in that: The following steps are involved: 1) Prepare an Au film on top of the first CVDF film; 2) Synthesize Ag film on the front and back of the second CVDF film; 3) Compounding the two films, with the Au film on top and the Ag film on the bottom, to form a composite sandwich nanofilm Au / Ag / Ag.

2. The preparation of the composite sandwich nanofilm Au / Ag / Ag as claimed in claim 1, characterized in that: In step 1), the thickness of the first CVDF film is 100 to 800 μm, and the thickness of the Au film is 20 to 180 nm.

3. The preparation of the composite sandwich nanofilm Au / Ag / Ag as claimed in claim 1, characterized in that: In step 2), the Ag film is prepared by glucose reduction method. First, ammonia water is added to the AgNO3 solution, and then the CVDF membrane is added and left to stand for activation and then placed in glucose solution for reduction reaction.

4. The preparation of the composite sandwich nanofilm Au / Ag / Ag as claimed in claim 3, characterized in that: The concentration of ammonia water is 0.1~0.6 M.

5. Composite sandwich nanofilm Au / Ag / Ag, characterized by: The method is prepared by any one of claims 1 to 4.

6. Use of the composite sandwich nanofilm Au / Ag / Ag according to claim 5 in SERS detection of netilmicin.

7. The use according to claim 6, characterized in that: The composite sandwich nanofilm Au / Ag / Ag was used as the SERS substrate. AgNPs sol and netilmicin solution were dripped on the upper film and then NaCl solution was added to mix them. SERS detection was performed at the optimal excitation wavelength.

8. The use according to claim 7, characterized in that: As the concentration of the sample solution increases, the Raman peak of the sample to be tested at a specific wavelength gradually increases. A standard curve is established using the relationship between the intensity of the Raman characteristic peak of the sample to be tested and the amount of the sample to be tested. The sample to be tested is then quantitatively analyzed and tested, and netilmicin is distinguished from similar aminoglycoside antibiotics based on the different Raman characteristic peaks.

9. The use according to claim 7, characterized in that: The optimal excitation wavelength refers to the 785 nm excitation wavelength; the Raman peak of netilmicin at a specific wavelength refers to the Raman peak of netilmicin at 1697 cm -1 The characteristic Raman peak at .

10. The use according to claim 7, characterized in that: The linear detection range of netilmicin is 10 -5 ~10 -9 M, detection limit is 4.35×10 -10 M, the relative standard deviation of SERS intensity is less than 10%.

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