A surface-enhanced Raman scattering detection method for netimi stars

Abstract

This invention provides a surface-enhanced Raman scattering (SERS) detection method for netilmicin, belonging to the field of analytical detection technology. The invention utilizes a mixture of silver nanowire solution, dichloromethane, and tetrabutylammonium nitrate solution, followed by liquid-liquid self-assembly to form a silver nanowire interface film. This film is then scraped off using a silicon wafer, and a silver nanoparticle solution is dropped onto the surface of the silver nanowire interface film on the silicon wafer, resulting in a silicon wafer with a silver nanowire-silver nanoparticle composite silver film on its surface. This wafer is used as a "hotspot" enhanced SERS substrate, enabling simple, low-cost, accurate, and highly sensitive quantitative analysis of netilmicin. The SERS signal of netilmicin is captured by AgNPs, and the silver nanowire-silver nanoparticle composite silver film is supported on the silicon wafer to enhance the SERS signal of netilmicin and lower the detection limit.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a surface-enhanced Raman scattering detection method for netilmic stars. Background Technology

[0002] Netilmicin, a semi-synthetic aminoglycoside antibiotic, is widely used clinically to treat serious infections caused by various drug-resistant Gram-negative bacteria, such as Pseudomonas aeruginosa and Klebsiella pneumoniae, due to its broad-spectrum antibacterial activity. These infections include sepsis, pneumonia, and complicated urinary tract infections. However, this drug is chemically stable and highly water-soluble, making it difficult to be naturally degraded in the environment or within organisms. Its accumulation in environmental media (such as water and soil) may not only exacerbate the spread of bacterial resistance but also enter the human body through the food chain. Long-term low-dose exposure to residual netilmicin may induce adverse reactions such as ototoxicity and nephrotoxicity, posing a potential threat to human health. Therefore, establishing an analytical method to accurately monitor netilmicin residues in environmental and biological samples is crucial.

[0003] Currently, the mainstream detection technologies for netilmicin mainly include high-performance liquid chromatography-fluorescence detection (HPLC-fluorescence), liquid chromatography-mass spectrometry (LC-MS), and microbial assays. HPLC-fluorescence detection typically requires derivatization of the drug to enhance the detection signal, but this process is cumbersome and the derivatization efficiency may affect the accuracy of the results. While LC-MS offers extremely high sensitivity and specificity, its equipment is expensive, operation is complex, and sample pretreatment requirements are extremely stringent, making rapid deployment in grassroots laboratories or field settings difficult. Microbial assays, although lower in cost, have long detection cycles, poor specificity, and are susceptible to interference from other antimicrobial components in the sample. In summary, existing methods have limitations in terms of sensitivity, specificity, and portability, and cannot fully meet the urgent need for rapid, on-site, and highly sensitive monitoring of netilmicin. Developing a new, simple, low-cost, and reliable rapid on-site detection method has become an important research direction in the field of environmental and food safety monitoring. Summary of the Invention

[0004] The purpose of this invention is to provide a surface-enhanced Raman scattering detection method for netilmic stars. The method provided by this invention is simple to operate, low in cost, and has high accuracy and sensitivity. It does not require complex operating techniques and can meet the requirements of large-scale and rapid analysis and detection.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a surface-enhanced Raman scattering detection method for netilmic stars, comprising the following steps: (1) A solution of silver nanoparticles was synthesized using trisodium citrate and ascorbic acid. (2) A two-step polyol addition method was used to synthesize silver nanowire solution; (3) Add the silver nanowire solution obtained in step (2) to dichloromethane, and then add tetrabutylammonium nitrate solution to perform liquid-liquid self-assembly to form a silver nanowire interface film. Then, scrape out the silver nanowire interface film with a silicon wafer, and then drop the silver nanoparticle solution obtained in step (1) onto the surface of the silver nanowire interface film on the silicon wafer to obtain a silicon wafer with a surface of silver nanowire-silver nanoparticle composite silver film. (4) Using the silicon wafer with a surface of silver nanowire-silver nanoparticle composite silver film obtained in step (3) as the SERS substrate, the concentration of netilmicin in the sample to be tested was detected by standard curve method combined with surface-enhanced Raman scattering detection.

[0006] Preferably, the silver nanoparticles in the silver nanoparticle solution in step (1) have a particle size of 30~70 nm.

[0007] Preferably, step (1) uses trisodium citrate and ascorbic acid to synthesize silver nanoparticle solution, which includes the following steps: mixing silver nitrate solution and ultrapure water, heating to reflux, adding a mixture containing trisodium citrate and ascorbic acid, and performing a reflux reaction to obtain silver nanoparticle solution.

[0008] Preferably, the silver nanowires in the silver nanowire solution in step (2) have a particle size of 2~10μm.

[0009] Preferably, step (3) uses a two-step dropwise polyol method to synthesize silver nanowire solution, which includes the following steps: heating the ethylene glycol solution of polyvinylpyrrolidone, adding the ethylene glycol solution of ferric chloride, stirring for the first time, adding the ethylene glycol solution of sodium chloride, stirring for the second time, adding the ethylene glycol solution of silver nitrate, stirring for the third time, and obtaining the silver nanowire solution.

[0010] Preferably, the concentration of the tetrabutylammonium nitrate solution in step (3) is 10. -3 ~10 -5 M.

[0011] Preferably, in step (3), the volume ratio of the silver nanowire solution, dichloromethane, tetrabutylammonium nitrate solution and silver nanoparticle solution is (0.6~5) mL: (1~5) mL: (100~500) μL: (5~20) μL.

[0012] Preferably, in step (4), the silicon wafer with a surface of silver nanowire-silver nanoparticle composite silver film is used as the SERS substrate, and the concentration of netilmicin in the sample is detected by standard curve method combined with surface-enhanced Raman scattering detection, including the following steps: Prepare standard solutions of netilmicin with gradient concentrations; Using the silicon wafer with a silver nanowire-silver nanoparticle composite silver film obtained in step (3) as the SERS substrate, the netilmicin standard solution was dropped onto the surface of the silver nanowire-silver nanoparticle composite silver film on the silicon wafer, and surface-enhanced Raman spectroscopy was performed. The netilmicin standard solution was measured to be at 931 cm⁻¹. -1 The characteristic Raman peak intensity at the specified location is used as the ordinate, and the logarithm of the concentration of the corresponding netilmicin standard solution is used as the abscissa to establish a standard curve and obtain a linear regression equation. Under the same detection conditions as the netilmicin standard solution, the sample to be tested was subjected to surface-enhanced Raman spectroscopy, and the sample was measured to be at 931 cm⁻¹. -1 The intensity of the characteristic Raman peak at a given location is substituted into the linear regression equation to calculate the concentration of netilmicin in the sample.

[0013] Preferably, the concentration of netilmicin in the netilmicin standard solution is 10. -5 ~10 -10 M.

[0014] Preferably, the detection conditions for surface-enhanced Raman detection of the netilmicin standard solution are as follows: the excitation wavelength is 785 nm, the volume ratio of the silver nanoparticle solution obtained in step (1) to the netilmicin standard solution is 1:(0.25~4), the mixing time of the silver nanowire-silver nanoparticle composite silver film and the netilmicin standard solution is 8~20s, and the pH value of the mixture formed by the silver nanowire-silver nanoparticle composite silver film and the netilmicin standard solution is 5~9.

[0015] This invention provides a surface-enhanced Raman scattering (SERS) method for detecting netilmicin. First, high-quality silver nanoparticle solutions and silver nanowire solutions are prepared separately. Then, the silver nanowire solution is mixed with dichloromethane and tetrabutylammonium nitrate solutions, and liquid-liquid self-assembly is performed to form a silver nanowire interface film. This film is then scraped off using a silicon wafer. Next, silver nanoparticle solution is dropped onto the surface of the silver nanowire interface film on the silicon wafer to form a silver nanowire-silver nanoparticle composite silver film. This yields a silicon wafer with a surface of the silver nanowire-silver nanoparticle composite silver film. Finally, the prepared surface plasmon resonance coupling... A composite silver nanowire-silver nanoparticle (AgNWs-AgNPs) film, serving as a "hotspot" enhanced SERS substrate, enables simple, low-cost, accurate, and highly sensitive quantitative analysis of netilmicin. It offers advantages such as simplicity, convenience, high controllability, and high detection efficiency. The method captures the SERS signal of netilmicin using AgNPs, and the silver nanowire-silver nanoparticle composite film is mounted on a silicon wafer to enhance the SERS signal of netilmicin and lower the detection limit. The linear detection range for netilmicin provided by this invention is 10... -5 ~10 -10 M, detection limit is 7.52×10 -11 The relative standard deviation of M and SERS intensity is less than 10%, requiring no complex operating techniques and meeting the requirements for large-scale and rapid analysis and detection. Attached Figure Description

[0016] Figure 1 This is a characterization diagram of the particle size and shape of silver nanoparticles in the silver nanoparticle solution prepared in Example 1 of the present invention. Figure 1 Image A is a SEM image of silver nanoparticles; Figure 1 Image B is a TEM image of a single silver nanoparticle; Figure 2 This is a characterization diagram of the particle size and shape of silver nanowires in the silver nanowire solution prepared in Example 1 of the present invention. Figure 2 Image A in the image is a SEM image of silver nanowires. Figure 2 Image B is a TEM image of a single silver nanowire; Figure 3 The silver nanoparticles (AgNPs) and silver nanowires (AgNWs) prepared in Example 1 of this invention, as well as a mixture of silver nanoparticles (AgNPs) with silver nanowire solutions and silver nanoparticle solutions (abbreviated as AgNPs+AgNWs), were respectively reacted with a concentration of 10 -5 The UV spectrum of the mixed netilmicin solution of M, where... Figure 3 In the middle, A represents the UV spectra of AgNPs and AgNWs. Figure 3 In the middle, B represents AgNPs and AgNPs+AgNWs, respectively, with a concentration of 10 -5UV spectrum of M netilmicin solution after mixing; Figure 4 The images show the SERS spectra of netilmicin solutions at different volume ratios, pH values, and mixing times according to the present invention. Figure 4 The SERS spectra in section A are for different volume ratios of AgNPs+AgNWs composite silver films and netilmicin solutions. Figure 4 B represents the concentration of 10 measured under different pH conditions. -5 SERS spectrum of netilmicin solution of M Figure 4 The middle image shows the SERS spectra of the CAgNPs+AgNWs composite silver film and the netilmicin solution at different mixing times. Figure 5 The SERS spectra of netilmicin obtained from ten parallel acquisitions in this invention and the 931 cm⁻¹ spectrum. -1 A histogram of SERS peak intensities, where, Figure 5 A in the diagram represents the SERS spectrum. Figure 5 The middle B is 931 cm -1 A bar chart showing the intensity of the SERS peak; Figure 6 The SERS spectra and standard curves of netilmicin standard solutions at different concentrations obtained in Example 1 of this invention are shown below. Figure 6 A in the diagram represents the SERS spectrum. Figure 6 B in the figure represents the established standard curve. Figure 7 The above are surface-enhanced Raman spectra of different aminoglycoside antibiotics of the present invention: netilmicin, streptomycin, tobramycin, sisomicin, neomycin, etilmicin, and spectinomycin. Detailed Implementation

[0017] This invention provides a surface-enhanced Raman scattering detection method for netilmic stars, comprising the following steps: (1) A solution of silver nanoparticles was synthesized using trisodium citrate and ascorbic acid. (2) A two-step polyol addition method was used to synthesize silver nanowire solution; (3) Add the silver nanowire solution obtained in step (2) to dichloromethane, and then add tetrabutylammonium nitrate solution to perform liquid-liquid self-assembly to form a silver nanowire interface film. Then, scrape out the silver nanowire interface film with a silicon wafer, and then drop the silver nanoparticle solution obtained in step (1) onto the surface of the silver nanowire interface film on the silicon wafer to obtain a silicon wafer with a surface of silver nanowire-silver nanoparticle composite silver film. (4) Using the silicon wafer with a surface of silver nanowire-silver nanoparticle composite silver film obtained in step (3) as the SERS substrate, the concentration of netilmicin in the sample to be tested was detected by standard curve method combined with surface-enhanced Raman scattering detection.

[0018] Unless otherwise specified, all raw materials used in this invention are commercially available products in the art.

[0019] In this invention, the particle size of the silver nanoparticles in the silver nanoparticle solution is preferably 30-70 nm, more preferably 40-60 nm. This invention controls the particle size of the silver nanoparticles in the solution to be within the 30-70 nm range, which serves as a "golden zone" for electromagnetic field enhancement, effectively exciting the strongest LSPR and achieving efficient signal amplification. Because the nanoparticles within this size range are much smaller than the laser wavelength, they can interact with the light field in a near-perfect dipole manner, maximizing the enhancement efficiency of Raman scattering. This avoids the situation where the dipole moment enhancement effect of a single particle is significantly insufficient due to excessively small particle size (<30 nm), resulting in limited overall signal strength. Simultaneously, it avoids the situation where the enhancement efficiency begins to decrease due to excessively large particle size (>70 nm). While larger particles can provide more adsorption surface, the enhancement efficiency of each particle will decrease significantly due to severe phase difference effects. Furthermore, the distribution of large particles is difficult to control and can easily lead to poor signal reproducibility.

[0020] In this invention, the method of synthesizing silver nanoparticle solution using trisodium citrate and ascorbic acid preferably includes the following steps: mixing silver nitrate solution and ultrapure water, heating to reflux, adding a mixture containing trisodium citrate and ascorbic acid, and carrying out a reflux reaction to obtain silver nanoparticle solution.

[0021] In this invention, the concentration of silver nitrate is preferably 0.01~0.05M. In this invention, the volume ratio of the silver nitrate solution to ultrapure water is preferably (1~5):(80~120). In this invention, the mass ratio of trisodium citrate, ascorbic acid, and ultrapure water in the mixture containing trisodium citrate and ascorbic acid is preferably (50~120) mg:(200~600) mg:(300~600) mL. In this invention, the stirring speed of the reflux reaction is preferably 1000~1400 r / min. In this invention, the temperature of the reflux reaction is preferably 60~90℃; the time of the reflux reaction is preferably 40~100 min, more preferably 60~90 min. By controlling the temperature and time of the reflux reaction within the above ranges, this invention can stably obtain silver nanoparticles of 30~70 nm, meeting the size requirements of SERS substrates. To ensure the monodispersity and stability of silver nanoparticles and avoid secondary nucleation or overgrowth, the particle size distribution is narrow, and the colloidal solution is not prone to sedimentation during long-term storage. The uniformly sized silver nanoparticles exhibit the strongest LSPR absorption and SERS enhancement effect at a specific excitation wavelength, and have good surface plasmon resonance performance. The fixed temperature and time window ensures that the synthesis results of different batches are highly consistent, which is conducive to the preparation of standardized SERS substrates and has good batch reproducibility.

[0022] In this invention, the particle size of the silver nanowires in the silver nanowire solution is preferably 2-10 μm, more preferably 3-7 nm. By controlling the particle size of the silver nanowires in the silver nanoparticle solution within the above range, this invention allows the silver nanowires to easily form a three-dimensional porous network, providing numerous nanowire intersections or end gaps. These locations are the main source of SERS "hot spots," which can greatly enhance the local electromagnetic field. Silver nanowires with particle sizes within the above range can form a dense and uniform hot spot array, with an enhancement factor reaching 10. 6 The above-mentioned enhancement effect, along with controlling the particle size within the aforementioned range, ensures the stability and reproducibility of silver nanowires, exhibiting good particle size / size uniformity and minimal batch-to-batch variation, which is beneficial for standardized SERS detection. Silver nanowires with particle sizes within the above range can be fabricated into large-area, flexible SERS substrates, increasing detection throughput and making them suitable for rapid on-site detection or microfluidic chip integration. In this invention, the concentration of silver nanowires in the silver nanowire solution is 1~5 mg / mL.

[0023] In this invention, the two-step polyol addition method for synthesizing silver nanowire solution preferably includes the following steps: heating a polyvinylpyrrolidone ethylene glycol solution, adding a ferric chloride ethylene glycol solution, stirring for the first time, adding a sodium chloride ethylene glycol solution, stirring for the second time, and then adding a silver nitrate ethylene glycol solution, stirring for the third time, to obtain the silver nanowire solution.

[0024] In this invention, the ethylene glycol solution of polyvinylpyrrolidone is preferably heated to 140-170°C, more preferably 150-160°C. In this invention, the concentration of polyvinylpyrrolidone in the ethylene glycol solution of polyvinylpyrrolidone is preferably 0.3-0.8 M. In this invention, the concentration of ferric chloride in the ethylene glycol solution of ferric chloride is preferably 0.03-0.08 M. In this invention, the concentration of sodium chloride in the ethylene glycol solution of sodium chloride is preferably 0.13-0.18 M. In this invention, the concentration of silver nitrate in the ethylene glycol solution of silver nitrate is preferably 0.13-0.18 M. In this invention, the preferred volume ratio of the ethylene glycol solution of polyvinylpyrrolidone, ethylene glycol solution of ferric chloride, ethylene glycol solution of sodium chloride, and ethylene glycol solution of silver nitrate is (40~50) mL : (100~200) μL : (100~200) μL : (10~20) mL. This invention controls the concentration and volume ratio of the ethylene glycol solutions of polyvinylpyrrolidone, ferric chloride, sodium chloride, and silver nitrate within the above range to ensure that the relative amounts of silver source, reducing agent, morphology control agent, and etching agent are at the optimal window, and that the ratio of PVP to Ag... +The molar ratio is reasonable, providing sufficient surface modification without hindering linear growth due to excessive PVP; simultaneously, the addition of trace amounts of FeCl3 and NaCl effectively controls the morphology without introducing excessive impurities or over-etching. In this invention, the first stirring time is preferably 2-10 min. In this invention, the second stirring time is preferably 2-10 min. In this invention, the third stirring time is preferably 2-3 h. This invention, based on a two-step dropwise polyol method using AgNO3 precursor and FeCl3 as a reaction promoter, can efficiently synthesize high-yield silver nanowires.

[0025] In this invention, the concentration of the tetrabutylammonium nitrate solution is preferably 10. -3 ~10 -5 M. In this invention, the preferred volume ratio of the silver nanowire solution, dichloromethane, tetrabutylammonium nitrate solution, and silver nanoparticle solution is (0.6~5) mL:(1~5) mL:(100~500) μL:(5~20) μL, more preferably (1~3) mL:(1.5~3) mL:(180~260) μL:(8~16) μL. This invention controls the volume ratio of the silver nanowire solution, dichloromethane, tetrabutylammonium nitrate solution, and silver nanoparticle solution within the above range to improve film uniformity. The optimized volume ratio avoids nanowire entanglement or particle aggregation, forming a single-layer or double-layer uniform film without macroscopic cracks or thick spots; it ensures uniform hotspot distribution, with silver nanowire intersections (line-to-line hotspots) and silver nanoparticle-nanowire gaps (point-to-line hotspots) working synergistically to maximize the density of "hotspots" and ensure uniform spatial distribution, allowing the SERS enhancement factor to stably reach 10. 7 The above measures improve signal reproducibility, controlling the RSD of different sites within a batch and between batches to within 10%, meeting quantitative detection requirements. They also enhance the stability of the prepared substrate; the appropriate amount of tetrabutylammonium nitrate can shield electrostatic repulsion, ensuring the assembled film firmly adheres to the hydrophobic or hydrophilic substrate surface and is less prone to detachment. In this invention, the silicon wafer is preferably a hydrophilic silicon wafer. This invention utilizes the hydrophilic silicon wafer to adsorb the hydrophilic ends of the silver nanowire interface film in the aqueous phase. After being scraped off by the hydrophilic silicon wafer, the silver nanowire interface film is transferred to the water-air interface. A silver nanoparticle solution is then dropped onto the surface of the silver nanowire interface film to form a silver nanowire-silver nanoparticle composite silver film, abbreviated as AgNWs-AgNPs composite silver film, ultimately yielding a silicon wafer with a surface of the silver nanowire-silver nanoparticle composite silver film.

[0026] In this invention, the silicon wafer with a surface of silver nanowire-silver nanoparticle composite silver film is used as the SERS substrate, and the concentration of netilmicin in the sample is detected by standard curve method combined with surface-enhanced Raman scattering detection. Preferably, the method includes the following steps: Prepare standard solutions of netilmicin with gradient concentrations; Using the silicon wafer with a silver nanowire-silver nanoparticle composite silver film obtained in step (3) as the SERS substrate, the netilmicin standard solution was dropped onto the surface of the silver nanowire-silver nanoparticle composite silver film on the silicon wafer, and surface-enhanced Raman spectroscopy was performed. The netilmicin standard solution was measured to be at 931 cm⁻¹. -1 The characteristic Raman peak intensity at the specified location is used as the ordinate, and the logarithm of the concentration of the corresponding netilmicin standard solution is used as the abscissa to establish a standard curve and obtain a linear regression equation. Under the same detection conditions as the netilmicin standard solution, the sample to be tested was subjected to surface-enhanced Raman spectroscopy, and the sample was measured to be at 931 cm⁻¹. -1 The intensity of the characteristic Raman peak at a given location is substituted into the linear regression equation to calculate the concentration of netilmicin in the sample.

[0027] In this invention, the concentration of netilmicin in the netilmicin standard solution is preferably 10. -5 ~10 -10 M.

[0028] In this invention, the detection conditions for surface-enhanced Raman spectroscopy (SERS) of the netilmicin standard solution are as follows: excitation wavelength of 785 nm, volume ratio of the silver nanoparticle solution to the netilmicin standard solution of 1:(0.25~4), mixing time of the silver nanowire-silver nanoparticle composite film and the netilmicin standard solution of 8~20 s, and pH value of the mixture formed by the silver nanowire-silver nanoparticle composite film and the netilmicin standard solution of 5~9. In this invention, the volume ratio of the silver nanoparticle solution to the netilmicin standard solution is more preferably 1:(0.8~2). In this invention, the mixing time of the silver nanowire-silver nanoparticle composite film and the netilmicin standard solution is more preferably 8~20 s. In this invention, the pH value of the mixture formed by the silver nanowire-silver nanoparticle composite film and the netilmicin standard solution is more preferably 6~8. This invention controls the detection conditions and parameters of surface-enhanced Raman spectroscopy (SERS) within the above-mentioned ranges, so that the SERS intensity of netilmicin reaches its maximum, thereby improving the accuracy and sensitivity of the detection.

[0029] The silver nanowire-silver nanoparticle composite silver film prepared by this invention has the characteristics of high sensitivity, high selectivity and repeatability, and the composite silver film has a good enhancement effect, which can construct a "hot spot" enhanced SERS substrate.

[0030] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0031] Unless otherwise specified, all experiments were repeated three times, and the results are expressed as averages.

[0032] Example 1 A surface-enhanced Raman scattering detection method for netilmic stars, comprising the following steps: (1) Take 5 mL of 0.01 M AgNO3 solution and add it to a flask containing 100 mL of ultrapure water. Stir and heat to boiling reflux state. Add 10 mL of mixed solution containing sodium citrate and ascorbic acid until the solution becomes a slightly green opaque liquid. Continue heating and refluxing at 80 °C for 60 min. After stopping heating, let it cool naturally to room temperature, seal and store in the dark to obtain silver nanoparticle sol with uniform particle size, i.e., silver nanoparticle solution. The volume ratio of the silver nitrate solution to ultrapure water is 5:100; the mass ratio of trisodium citrate, ascorbic acid, and ultrapure water in the mixture containing trisodium citrate and ascorbic acid is 100mg:500mg:500mL; the particle size of the silver nanoparticles is 51.08±4.17 nm. (2) Using ethylene glycol solution (EG) as solvent, polyvinylpyrrolidone (PVP), ferric chloride (FeCl3), sodium chloride (NaCl) and silver nitrate (AgNO3) were dissolved in EG for at least 2 h to prepare ethylene glycol solutions of polyvinylpyrrolidone, ferric chloride, sodium chloride and silver nitrate respectively. At 25℃, 40 mL of 0.56 M PVP ethylene glycol solution was added to a 250 mL flask and stirred until the temperature stabilized at 160℃. Then, 100 μL of 0.05 M FeCl3 ethylene glycol solution was added and stirred for 5 minutes. Next, 100 μL of 0.15 M NaCl ethylene glycol solution was added and stirred for 5 minutes. Finally, 10 mL of 0.15 M AgNO3 ethylene glycol solution was added and stirred for 2 hours to obtain a silver nanowire solution. The volume ratio of the ethylene glycol solution of polyvinylpyrrolidone, the ethylene glycol solution of ferric chloride, the ethylene glycol solution of sodium chloride, and the ethylene glycol solution of silver nitrate is 40 mL: 100 μL: 100 μL: 10 mL; the particle size of the silver nanowires in the silver nanowire solution is 5 ± 0.77 μm; the concentration of the silver nanowires in the silver nanowire solution is 3.3 mg / mL. (3) Add 2 mL of the silver nanowire solution obtained in step (2) to a test tube containing 2 mL of dichloromethane, and then add 200 μL of a solution with a concentration of 10 -5 M tetrabutylammonium nitrate solution is used to perform liquid-liquid self-assembly to form a silver nanowire interface film. Then, the silver nanowire interface film is scraped out with a hydrophilic silicon wafer. Then, 10 μL of silver nanoparticle solution obtained in step (1) is dropped onto the surface of the silver nanowire interface film on the silicon wafer to obtain a silicon wafer with a surface of silver nanowire-silver nanoparticle composite silver film. In this process, the hydrophilic silicon wafer adsorbs the hydrophilic end of the silver nanowire interface film in the aqueous phase, and after being scraped out by the hydrophilic silicon wafer, the silver nanowire interface film is transferred to the water-air interface. Then, a silver nanoparticle solution is dropped onto the surface of the silver nanowire interface film to form a silver nanowire-silver nanoparticle composite silver film, which is simply referred to as AgNWs-AgNPs composite silver film. The volume ratio of the silver nanowire solution, dichloromethane, tetrabutylammonium nitrate solution, and silver nanoparticle solution is 2 mL: 2 mL: 200 μL: 10 μL; (4) Prepared using ultrapure water as solvent, with concentrations of 10... -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M and 10 -10 M netilmicin standard solution; Using the silicon wafer with a surface of silver nanowire-silver nanoparticle composite silver film obtained in step (3) as a SERS substrate, the netilmicin standard solution was dropped onto the surface of the silver nanowire-silver nanoparticle composite silver film on the silicon wafer, and surface-enhanced Raman spectroscopy was performed. The concentration was measured to be 10. -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M and 10 -10 M netilmicin standard solution at 931 cm -1 The characteristic Raman peak intensities at the specified locations were 1.1 W, 0.8 W, 0.6 W, 0.5 W, 0.4 W, and 0.26 W, respectively. A standard curve was established with the characteristic Raman peak intensities as the ordinate (y) and the logarithm of the concentration of the corresponding netilmicin standard solution as the abscissa (x). The linear regression equation was obtained as y = 1517.6 x + 18155.58. Under the same detection conditions as the netilmicin standard solution, the sample was subjected to surface-enhanced Raman spectroscopy, and the sample was measured to be at 931 cm⁻¹. -1The characteristic Raman peak intensity at the specified location is 1.1 W. Substituting this into the linear regression equation, the concentration of netilmicin in the sample was calculated to be 7.52 × 10⁻⁶. -11 M; The detection conditions for surface-enhanced Raman spectroscopy of the netilmicin standard solution were as follows: excitation wavelength of 785 nm, volume ratio of silver nanoparticle solution to netilmicin standard solution of 1:1, mixing time of silver nanowire-silver nanoparticle composite film and netilmicin standard solution of 10 s, and pH value of the mixture formed by silver nanowire-silver nanoparticle composite film and netilmicin standard solution of 6.

[0033] The particle size and shape of the silver nanoparticles in the silver nanoparticle solution prepared in Example 1 were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The particle size and shape characterization images of the silver nanoparticles in the silver nanoparticle solution prepared in Example 1 are shown below. Figure 1 As shown, where, Figure 1 Image A is a SEM image of silver nanoparticles; Figure 1 Image B is a TEM image of a single silver nanoparticle. (Source: [Insert image here]) Figure 1 It can be seen that the silver nanoparticles prepared in Example 1 have a particle size of 51.08±4.17 nm and an appearance that is nearly round, but slightly elliptical.

[0034] The particle size and shape of the silver nanowires in the silver nanowire solution prepared in Example 1 were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The particle size and shape characterization images of the silver nanowires in the silver nanowire solution prepared in Example 1 are shown below. Figure 2 As shown, where, Figure 2 Image A in the image is a SEM image of silver nanowires. Figure 2 Image B is a TEM image of a single silver nanowire. Figure 2 It can be seen that the silver nanowires prepared in Example 1 have a particle size of 5±0.77 μm and an appearance of elongated strips.

[0035] Silver nanoparticles (AgNPs) and silver nanowires (AgNWs) prepared in Example 1, as well as a mixture of silver nanoparticles (AgNPs) with silver nanowire solutions and silver nanoparticle solutions (abbreviated as AgNPs+AgNWs), were respectively reacted with a concentration of 10 -5 The UV spectrum of the mixed netilmicin solution of M, where... Figure 3 In the image, A represents the UV spectra of AgNPs and AgNWs, derived from... Figure 3 As can be seen from A, AgNWs successfully synthesized; Figure 3 In this context, B represents a mixture of silver nanoparticles (AgNPs) with the silver nanowire solution and the silver nanoparticle solution (abbreviated as AgNPs+AgNWs), respectively, with a concentration of 10...-5 The UV spectrum of the mixed netilmicin solution of M, by Figure 3 As shown in B, the ultraviolet peak shifts after AgNPs and AgNWs are mixed, indicating that there is an interaction between AgNPs+AgNWs and netilmicin.

[0036] Investigation 1: The optimal volume ratio of silver nanoparticle solution to netilmicin solution The concentration of the AgNPs-AgNWs composite silver film was fixed, and the concentration of the netilmicin solution was 10. -5 M; The volume ratios of the silver nanoparticle solution and the netilmicin solution were adjusted to 1:0.25, 1:0.5, 1:1, 1:2, and 1:4, respectively. SERS spectra were acquired according to the method in Example 1, and the SERS spectra at different volume ratios of silver nanoparticle solution and netilmicin solution were obtained as follows. Figure 4 As shown in Figure A. From Figure 4 As can be seen from Figure A, the SERS intensity of the netilmicin solution is strongest when the volume ratio of the silver nanoparticle solution to the netilmicin solution reaches 1:1. Therefore, during the detection process, the preferred volume ratio of the silver nanoparticle solution to the netilmicin solution is 1:(0.25~4), and the most preferred ratio is 1:1.

[0037] Investigation 2: Optimal pH value of the mixture formed by AgNPs-AgNWs composite silver film and netilmicin solution Fixed AgNPs-AgNWs composite silver film with a concentration of 10 -5 The volume ratio of netilmicin solution M to the total solution was 1:1. The pH of the mixture formed by the AgNPs-AgNWs composite silver film and netilmicin solution was adjusted to 2, 4, 6, 8, and 10, respectively. SERS spectra were collected according to the method in Example 1, and the concentrations measured under different pH conditions were 10. -5 The SERS spectrum of netilmicin solution M is as follows: Figure 4 As shown in B. From Figure 4 As can be seen from Figure B, the intensity of the surface-enhanced Raman signal peak of netilmicin is relatively strong when the pH value is 5-9, and the intensity of the surface-enhanced Raman signal peak of netilmicin is strongest at pH=6. Therefore, the optimal pH value for the detection system of netilmicin is 6.

[0038] Investigation 3: Optimal mixing time of AgNPs-AgNWs composite silver film and netilmicin With a fixed concentration of the AgNPs-AgNWs composite silver film, the concentration of the netilmicin solution was 10. -5M, the volume ratio of the two is 1:1, and the pH of the mixture is 6; the mixing time of AgNPs-AgNWs composite silver film and netilmicin solution is adjusted to 5s, 10s, 15s, 20s, and 25s, respectively, and SERS spectra are collected according to the method of Example 1. The SERS spectra of AgNPs-AgNWs composite silver film and netilmicin solution at different mixing times are shown below. Figure 4 As shown in C, by Figure 4 As can be seen from Figure C, when the mixing time of AgNPs-AgNWs composite silver film and netilmicin solution is 8~20s, the SERS intensity of netilmicin is relatively strong, and the SERS intensity of netilmicin reaches its strongest when the mixing time is 10s.

[0039] Investigation 4: Homogeneity of the SERS substrate of netilmicin Take AgNPs-AgNWs composite silver film and a concentration of 10 -5 A netilmicin solution of M was prepared at a fixed volume ratio of 1:1, with the pH adjusted to 6 and a mixing time of 10 s. SERS spectra were acquired according to the method in Example 1, and ten SERS spectra were randomly acquired on the substrate. The SERS peak intensities of the ten parallel acquisitions of netilmicin were compared to determine the homogeneity of the SERS substrate. The ten parallel acquisitions of netilmicin SERS spectra and a 931 cm⁻¹ peak intensity were obtained. -1 The bar chart of SERS peak intensity is as follows Figure 5 As shown, where, Figure 5 Image A shows the SERS spectrum of netilmicin measured in ten parallel measurements. Figure 5 B represents ten parallel measurements of netilmicin at 931 cm. -1 A histogram of SERS peak intensity. (From...) Figure 5 It can be seen that the relative standard deviation (RSD) calculated from the SERS characteristic peak intensities obtained from ten parallel detections is 931 cm⁻¹. -1 The concentration was 6.70%, all less than 10%, indicating that the SERS substrate has good homogeneity and can be applied to the quantitative analysis of netilmicin by surface-enhanced Raman spectroscopy.

[0040] Investigation 5: SERS detection of netilmicin The SERS spectra and standard curves of netilmicin standard solutions at different concentrations obtained in Example 1 are shown below. Figure 6 As shown.

[0041] in, Figure 6 Image A shows the SERS spectra of netilmicin standard solutions at different concentrations, with the concentrations from top to bottom being 10... -5 M, 10 -6 M, 10 -7 M, 10 -8M, 10 -9 M, 10 -10 M. By Figure 6 As can be seen from Figure A, the intensity of the surface-enhanced Raman peak of netilmicin increases with increasing concentration.

[0042] Figure 6 In Example 1, B represents the standard curve established after detecting different concentrations of netilmicin standard solutions. -5 ~10 -10 Within the range of M, netilmicin 931cm -1 The intensity of the characteristic peak showed a negative logarithmic relationship with the netilmicin concentration, but also exhibited a positive correlation, yielding a linear regression equation y = 1517.6x + 18155.58, R0. 2 = 0.9930, the limit of detection (LOD) is 7.52 × 10 -11 M.

[0043] Investigation 6: Selectivity of AgNPs+AgNWs composite silver membrane for netilmicin, streptomycin, tobramycin, sisomicin, neomycin, etilmicin, and spectinomycin Take AgNPs-AgNWs composite silver films and mix them with 10% AgNPs-AgNWs composite silver films. -5 M solutions of netilmicin, streptomycin, tobramycin, sisomicin, neomycin, etimicin, and spectinomycin were mixed at a fixed volume ratio of 1, and the pH was adjusted to 5. SERS spectra were acquired using an excitation wavelength of 785 nm. The surface-enhanced Raman spectra of different aminoglycoside antibiotics, including netilmicin, streptomycin, tobramycin, sisomicin, neomycin, etimicin, and spectinomycin, are shown below. Figure 7 As shown, from top to bottom, the drugs are netilmicin, streptomycin, tobramycin, sisomicin, neomycin, etilmicin, and spectinomycin. Figure 7 It can be seen that under weakly acidic conditions, the six antibiotics can be divided into three categories based on peak shape: (1) spectinomycin, (2) netilmicin, streptomycin, and etimicin, and (3) neomycin and tobramycin. Spectinomycin did not show a characteristic peak; for netilmicin, streptomycin, tobramycin, neomycin, and etimicin, tobramycin and neomycin showed a peak at 614 cm⁻¹. -1 The peak disappears, 931cm -1 The peak shifted. For netilmicin, streptomycin, and etilmicin, streptomycin showed a shift at 685 cm⁻¹. -1 There is a peak, and etimicin is at 1187cm. -1 There is a peak at 685cm. Netilmicin is at 685cm. -1 The peak position shifted at 1187cm. -1No peak was observed. Therefore, under weakly acidic conditions (pH=6), the AgNPs-AgNWs composite silver membrane, as a SERS substrate, can distinguish netilmicin, streptomycin, tobramycin, sisomicin, neomycin, etimicin, and spectinomycin, with good selectivity for netilmicin.

[0044] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A surface-enhanced Raman scattering detection method for netilmic stars, characterized in that, Includes the following steps: (1) A solution of silver nanoparticles was synthesized using trisodium citrate and ascorbic acid. (2) A two-step polyol addition method was used to synthesize silver nanowire solution; (3) Add the silver nanowire solution obtained in step (2) to dichloromethane, and then add tetrabutylammonium nitrate solution to perform liquid-liquid self-assembly to form a silver nanowire interface film. Then, scrape out the silver nanowire interface film with a silicon wafer, and then drop the silver nanoparticle solution obtained in step (1) onto the surface of the silver nanowire interface film on the silicon wafer to obtain a silicon wafer with a surface of silver nanowire-silver nanoparticle composite silver film. (4) Using the silicon wafer with a surface of silver nanowire-silver nanoparticle composite silver film obtained in step (3) as the SERS substrate, the concentration of netilmicin in the sample to be tested was detected by standard curve method combined with surface-enhanced Raman scattering detection.

2. The surface-enhanced Raman scattering detection method for netilmic stars according to claim 1, characterized in that, In step (1), the silver nanoparticles in the silver nanoparticle solution have a particle size of 30~70nm.

3. The surface-enhanced Raman scattering detection method for netilmic stars according to claim 1, characterized in that, The step (1) uses trisodium citrate and ascorbic acid to synthesize silver nanoparticle solution, which includes the following steps: mixing silver nitrate solution and ultrapure water, heating to reflux, adding a mixture containing trisodium citrate and ascorbic acid, and carrying out a reflux reaction to obtain silver nanoparticle solution.

4. The surface-enhanced Raman scattering detection method for netilmic stars according to claim 1, characterized in that, In step (2), the silver nanowires in the solution have a particle size of 2~10μm.

5. The surface-enhanced Raman scattering detection method for netilmic stars according to claim 1, characterized in that, The step (3) uses a two-step dropwise polyol method to synthesize silver nanowire solution, which includes the following steps: heating the ethylene glycol solution of polyvinylpyrrolidone, adding the ethylene glycol solution of ferric chloride, stirring for the first time, adding the ethylene glycol solution of sodium chloride, stirring for the second time, and then adding the ethylene glycol solution of silver nitrate, stirring for the third time to obtain the silver nanowire solution.

6. The surface-enhanced Raman scattering detection method for netilmic stars according to claim 1, characterized in that, The concentration of the tetrabutylammonium nitrate solution in step (3) is 10. -3 ~10 -5 M.

7. The surface-enhanced Raman scattering detection method for netilmic stars according to claim 6, characterized in that, In step (3), the volume ratio of the silver nanowire solution, dichloromethane, tetrabutylammonium nitrate solution and silver nanoparticle solution is (0.6~5) mL: (1~5) mL: (100~500) μL: (5~20) μL.

8. The surface-enhanced Raman scattering detection method for netilmic stars according to claim 1, characterized in that, In step (4), the silicon wafer with a surface of silver nanowire-silver nanoparticle composite silver film is used as the SERS substrate, and the concentration of netilmicin in the sample is detected by standard curve method combined with surface-enhanced Raman scattering detection. The steps include: Prepare standard solutions of netilmicin with gradient concentrations; Using the silicon wafer with a silver nanowire-silver nanoparticle composite silver film obtained in step (3) as the SERS substrate, the netilmicin standard solution was dropped onto the surface of the silver nanowire-silver nanoparticle composite silver film on the silicon wafer, and surface-enhanced Raman spectroscopy was performed. The netilmicin standard solution was measured to be at 931 cm⁻¹. -1 The characteristic Raman peak intensity at the specified location is used as the ordinate, and the logarithm of the concentration of the corresponding netilmicin standard solution is used as the abscissa to establish a standard curve and obtain a linear regression equation. Under the same detection conditions as the netilmicin standard solution, the sample was subjected to surface-enhanced Raman spectroscopy, and the sample was measured to be at 931 cm⁻¹. -1 The intensity of the characteristic Raman peak at a given location is substituted into the linear regression equation to calculate the concentration of netilmicin in the sample.

9. The surface-enhanced Raman scattering detection method for netilmic stars according to claim 8, characterized in that, The concentration of netilmicin in the netilmicin standard solution is 10. -5 ~10 -10 M.

10. The surface-enhanced Raman scattering detection method for netilmic stars according to claim 8, characterized in that, The detection conditions for surface-enhanced Raman spectroscopy of the netilmicin standard solution are as follows: the excitation wavelength is 785 nm, the volume ratio of the silver nanoparticle solution and the netilmicin standard solution obtained in step (1) is 1:(0.25~4), the mixing time of the silver nanowire-silver nanoparticle composite silver film and the netilmicin standard solution is 8~20 s, and the pH value of the mixture formed by the silver nanowire-silver nanoparticle composite silver film and the netilmicin standard solution is 5~9.

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