Method for detecting neomycin and apramycin in veterinary drugs

Abstract

The present application belongs to the technical field of veterinary drug residue detection, and particularly relates to a detection method of neomycin and apramycin in veterinary drugs, wherein a hydroxylated silver nanowire dispersion liquid and a dopamine modified Ag NCs@TiO2 nanoparticle dispersion liquid are mixed in equal volume, then embedded in a PDMS flexible substrate material to obtain a flexible SERS substrate material, a spiked sample solution is dropped onto the surface of the flexible SERS substrate material, and then dried and subjected to SERS detection, a linear regression equation is fitted by taking the characteristic peak intensity of the antibiotic and the logarithmic value of the concentration of the spiked sample solution as variables, a sample solution to be detected is dropped onto the surface of the flexible SERS substrate material, and then subjected to SERS detection to calculate the residual concentration of the antibiotic in the sample solution to be detected. The present application adopts a surface enhanced Raman scattering technology (SERS) to detect the residual amounts of neomycin and apramycin in meat products, and the detection is faster and simpler.

Description

Technical Field

[0001] This invention belongs to the field of veterinary drug residue detection technology, specifically relating to a method for detecting neomycin and aspirin in veterinary drugs. Background Technology

[0002] Aminoglycoside antibiotics are a class of broad-spectrum antibiotics composed of amino sugars and aminocyclic alcohols linked by an oxygen bridge. Due to their good antibacterial efficacy against both Gram-positive and Gram-negative infections, they are widely used in animal husbandry, aquaculture, and veterinary medicine. However, the misuse and overuse of aminoglycoside antibiotics have led to their residues in the environment and agricultural products causing nephrotoxicity, ototoxicity, and neuromuscular blockade in humans. Therefore, detection methods for aminoglycoside antibiotics have become a hot topic of research.

[0003] In the process of testing the quality of livestock products, the detection method for neomycin and apramycin generally adopts the microbial potency method. This method involves the diffusion of antibiotics in agar plates under strictly controlled agar culture conditions, forming transparent inhibition zones. The diameter of the inhibition zone is linearly related to the concentration (or potency) of the antibiotic within a specific range. By accurately measuring and comparing the size of the inhibition zones produced by a standard of known concentration and a test sample of unknown concentration, the potency of the test sample can be calculated.

[0004] However, this method is complex and time-consuming. The entire detection process includes multiple steps such as bacterial activation, culture medium preparation, culture, and inhibition zone measurement, which usually takes 16 to 24 hours or even longer. It cannot achieve rapid detection and has low efficiency. Furthermore, changes in any variable in multiple steps may interfere with the accuracy of the detection results. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting neomycin and aspirin in veterinary drugs, so as to solve the above-mentioned technical problems.

[0006] To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows: The detection method for neomycin and apramycin in veterinary drugs includes the following steps: S1. Sample pretreatment; Grind the frozen sample, add acetonitrile and acetic acid dropwise, and make up to 15 mL to obtain a mixed sample. Add anhydrous magnesium sulfate and sodium chloride to the mixed sample, vortex and centrifuge, and transfer the supernatant to a mixture of anhydrous magnesium sulfate and PSA adsorbent. Vortex and centrifuge, and take the supernatant as the sample solution to be tested. S2. Establishment of the standard curve; A blank sample solution was prepared from a blank sample that does not contain antibiotics according to the method in step S1; The 1 mol / L antibiotic standard stock solution was serially diluted with blank sample solution to obtain a series of spiked sample solutions. The spiked sample solution was dropped onto the surface of a flexible SERS substrate material, dried, and then subjected to SERS detection. A linear regression equation was fitted using the logarithm of the characteristic peak intensity of the antibiotic and the concentration of the spiked sample solution. S3. Drop the sample solution to be tested onto the surface of the flexible SERS substrate material, let it dry, and then perform SERS detection. Record the characteristic peak intensity of antibiotics in the SERS spectrum of the sample solution to be tested, substitute it into the linear regression equation, and calculate the residual concentration of antibiotics in the sample solution to be tested. The method for preparing the flexible SERS substrate material is as follows: A mixed dispersion was obtained by mixing equal volumes of hydroxylated silver nanowire dispersion and dopamine-modified Ag NCs@TiO2 nanoparticle dispersion. The PDMS prepolymer and crosslinking agent were mixed evenly, and the mixture was placed in a refrigerator to remove air bubbles, thus obtaining a crosslinking solution. Wipe the surface of the glass slide and wait for it to dry completely. Then, disperse the mixed dispersion on the glass slide and let it dry naturally at room temperature. Next, spread the crosslinking liquid on the glass slide and dry it. Finally, peel the film off the glass slide and cut it into small pieces to obtain the flexible SERS substrate material.

[0007] As a further improvement, the preparation method of the hydroxylated silver nanowire dispersion is as follows: Maltose monohydrate was added to deionized water and stirred thoroughly until completely dissolved to prepare a 0.12 mol / L reducing agent solution. Polyvinylpyrrolidone was dissolved in deionized water, and 0.06 mol / L silver nitrate solution and reducing agent solution were added. After stirring and mixing thoroughly, 0.06 mol / L sodium chloride solution was added dropwise, and stirring and mixing continued. The mixture was then heated to 160-165℃ and reacted for 18-20 hours. After cooling, the mixture was allowed to stand and separate into layers. The precipitate was washed three times with deionized water to obtain flexible silver nanowires. The flexible silver nanowires were then subjected to alkaline heat treatment and dispersed in ethanol to obtain a hydroxylated silver nanowire dispersion.

[0008] As a further improvement, the volume ratio of the silver nitrate solution, reducing agent solution, and sodium chloride solution is 1:1:0.8~1, and the mass-volume ratio of the polyvinylpyrrolidone and silver nitrate solution is 4g:1mL.

[0009] As a further improvement, the preparation method of dopamine-modified Ag NCs@TiO2 nanoparticle dispersion is as follows: Preparation of Ag NCs@TiO2 nanoparticles; Tetrabutyl titanate was added to ethanol under a nitrogen atmosphere and stirred to obtain a tetrabutyl titanate solution. Ag NCs were added to ethanol and dispersed evenly to obtain an Ag NCs solution; Ag NCs solution and dimethylamine solution were mixed, sonicated and stirred for 25-30 min, then tetrabutyl titanate solution was added dropwise, stirred for 1 h, heated to 160-165℃ and reacted for 10-12 h, cooled and centrifuged to separate the precipitate, washed and dried to obtain Ag NCs@TiO2 nanoparticles. Preparation of dopamine-modified Ag NCs@TiO2 nanoparticles; Ag NCs@TiO2 nanoparticles were added to water and mixed evenly. Then, hexamethylenetetramine was added and mixed evenly. Dopamine hydrochloride was added and the mixture was stirred at room temperature for 2-4 hours. After centrifugation, the precipitate was separated, washed, dried, and then redispersed in ethanol to obtain dopamine-modified Ag NCs@TiO2 nanoparticles.

[0010] As a further improvement, the preparation method of the Ag NCs is as follows: 0.18 mmol / L polyvinylpyrrolidone glycol solution is added to 3.0 mmol / L sodium sulfide glycol solution, and then 0.28 mol / L silver nitrate glycol solution is immediately added. After reacting for 10 min, the mixture is centrifuged to separate the precipitate, and the precipitate is washed to obtain Ag NCs.

[0011] As a further improvement, the volume ratio of the polyvinylpyrrolidone glycol solution, sodium sulfide glycol solution, and silver nitrate glycol solution is 15~18:1:5.

[0012] As a further improvement, in the preparation process of Ag NCs@TiO2 nanoparticles, the volume ratio of the dimethylamine solution to tetrabutyl titanate is 10:1, and the mass-volume ratio of Ag NCs to tetrabutyl titanate is 1 mg: 5 μL. In the preparation of dopamine-modified Ag NCs@TiO2 nanoparticles, the mass ratio of Ag NCs@TiO2 nanoparticles, hexamethylenetetramine, and dopamine hydrochloride is 10~11:13~13.5:1.

[0013] As a further improvement, in step S1, the volume fraction of acetic acid in the mixed sample is 0.5-1%, and the mass of the frozen sample is 10-12g.

[0014] As a further improvement, in the preparation method of the flexible SERS substrate material, the mass concentrations of the hydroxylated silver nanowire dispersion and the dopamine-modified Ag NCs@TiO2 nanoparticle dispersion are 5 mg / mL and 15 mg / mL, respectively; the mass ratio of the PDMS prepolymer to the crosslinking agent is 10:1; and the volume ratio of the mixed dispersion to the crosslinking liquid is 2:1.

[0015] As a further improvement, the antibiotic is neomycin sulfate or apramycin.

[0016] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows: This invention provides a method for detecting neomycin and apramycin in veterinary drugs. The method uses surface-enhanced Raman scattering (SERS) to detect the residues of these two veterinary drugs in meat products, making the detection faster and simpler.

[0017] In this invention, silver nanowires and Ag NCs@TiO2 nanoparticles are embedded in a polydimethylsiloxane (PDMS) substrate material in the prepared flexible SERS substrate material. Compared with the existing methods of depositing or adhering metal nanoparticles on the surface of flexible substrate materials, the metal nanoparticles in the flexible SERS substrate material of this invention are not easily detached, thus improving the repeatability and stability of the substrate material.

[0018] This invention obtains hydroxylated silver nanowires by thermal alkali treatment of silver nanowires, and obtains dopamine-modified Ag NCs@TiO2 nanoparticles by dopamine modification. In a mixed dispersion, the interaction between hydroxyl and amino groups causes the dopamine-modified Ag NCs@TiO2 nanoparticles to be uniformly distributed on the outer side of the hydroxylated silver nanowires. The TiO2 in the dopamine-modified Ag NCs@TiO2 nanoparticles not only exhibits Raman enhancement with the silver in the dopamine-modified Ag NCs@TiO2 nanoparticles, but also with the silver in the hydroxylated silver nanowires. The combined use of these two materials results in a higher Raman enhancement effect, further improving the detection sensitivity for antibiotics.

[0019] The Ag NCs@TiO2 nanoparticles prepared in this invention employ a cubic structure, which has sharp edges and apex corners, enabling stronger plasmon resonance and further improving the Raman enhancement effect, thereby enhancing the detection sensitivity for antibiotics. Attached Figure Description

[0020] Figure 1 This is a SEM image of the flexible silver nanowires in Embodiment 1 of the present invention;

[0021] Figure 2 This is a SEM image of Ag NCs in Embodiment 1 of the present invention;

[0022] Figure 3 This is a SEM image of the flexible SERS substrate material in Embodiment 1 of the present invention;

[0023] Figure 4 This is the standard curve of neomycin sulfate in Example 4 of this invention;

[0024] Figure 5 This is the standard curve of apramycin in Example 5 of the present invention. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0026] In this invention, polyvinylpyrrolidone is PVP-K30.

[0027] Example 1 This example provides a method for preparing a flexible SERS substrate material, specifically including the following steps:

[0028] S1. Preparation of flexible silver nanowires with high aspect ratio;

[0029] Add 0.0306 g of silver nitrate to 3 mL of deionized water, stir magnetically for 10 min until completely dissolved, store in the dark to obtain a 0.06 mol / L silver nitrate solution;

[0030] Add 0.1232 g of maltose monohydrate to 3 mL of deionized water and stir thoroughly for 10 min until completely dissolved to obtain a 0.12 mol / L reducing agent solution;

[0031] Add sodium chloride to 3 mL of deionized water and stir thoroughly for 10 min until completely dissolved to obtain a 0.06 mol / L sodium chloride solution;

[0032] Take 27 mL of deionized water, add 1.2 g of polyvinylpyrrolidone (PVP-K30), and stir magnetically for 30 min until completely dissolved. Then add silver nitrate solution and reducing agent solution, and continue stirring for 10 min to mix thoroughly. Then add sodium chloride solution dropwise, and continue stirring for 10 min. Then heat the reaction at 160℃ for 20 h. After cooling, a flexible silver nanowire solution is obtained. After standing and separating into layers, take the precipitate and add it to 6 mL of deionized water. Centrifuge at 3000 rpm for 5 min. Continue to take the precipitate, disperse it in deionized water and centrifuge. Repeat this process three times. The precipitate is the flexible silver nanowire.

[0033] like Figure 1The image shown is a scanning electron microscope (SEM) image of the flexible silver nanowires.

[0034] Preparation of S2, hydroxylated silver nanowire dispersion;

[0035] A 5 mg / mL hydroxylated silver nanowire dispersion was prepared by dispersing flexible silver nanowires in ethanol after alkaline heat treatment.

[0036] Specifically, the alkaline heat treatment involves adding flexible silver nanowires to a sodium hydroxide or potassium hydroxide solution, heating and stirring at 50-60°C for 10-13 hours, followed by centrifugation and washing. This method is existing technology.

[0037] Preparation of S3 and Ag NCs@TiO2 nanoparticles;

[0038] Ethylene glycol was stirred and heated at 150℃ for 1 h. After cooling, silver nitrate, sodium sulfide hydrate (Na2S·9H2O), and polyvinylpyrrolidone were added to 6 mL of ethylene glycol to obtain 0.28 mol / L silver nitrate ethylene glycol solution, 3.0 mmol / L sodium sulfide ethylene glycol solution, and 0.18 mmol / L polyvinylpyrrolidone ethylene glycol solution, respectively.

[0039] Add 100 μL of sodium sulfide ethylene glycol solution to the flask, wait for 8 min, then add 1.5 mL of polyvinylpyrrolidone ethylene glycol solution, and immediately add 0.5 mL of silver nitrate ethylene glycol solution. After reacting for 10 min, centrifuge at 9000 rpm for 10 min, take the precipitate, wash it 4 times with 50% ethanol solution, and collect Ag NCs;

[0040] like Figure 2 The image shown is a scanning electron microscope (SEM) image of Ag NCs.

[0041] Under a nitrogen atmosphere, 5 μL of tetrabutyl titanate was added to 5 mL of ethanol and stirred to obtain a tetrabutyl titanate solution.

[0042] 1 mg of Ag NCs was added to 2 mL of ethanol and dispersed evenly to obtain an Ag NCs solution;

[0043] Ag NCs solution and 50 μL dimethylamine solution (40% dimethylamine aqueous solution) were mixed, sonicated and stirred for 25 min, then tetrabutyl titanate solution was added dropwise, stirred for 1 h, heated to 160℃ and reacted for 12 h, cooled and centrifuged to separate the precipitate, washed three times with ethanol, and dried under vacuum at 60℃ to obtain Ag NCs@TiO2 nanoparticles;

[0044] S4. Preparation of dopamine-modified Ag NCs@TiO2 nanoparticle dispersion;

[0045] 0.153 g Ag NCs@TiO2 nanoparticles were added to 38 mL of water and mixed thoroughly. Then, 0.1989 g of hexamethylenetetramine was added and mixed thoroughly. Finally, 0.0153 g of dopamine hydrochloride was added and the mixture was stirred at room temperature for 2 h. After centrifugation at 10000 rpm for 2 min, the precipitate was separated, washed three times with ethanol, and air-dried to obtain dopamine-modified Ag NCs@TiO2 nanoparticles.

[0046] Dopamine-modified AgNCs@TiO2 nanoparticles were dispersed in ethanol to prepare a 15 mg / mL dopamine-modified AgNCs@TiO2 nanoparticle dispersion.

[0047] S5, Preparation of flexible SERS substrate materials;

[0048] An equal volume of hydroxylated silver nanowire dispersion with a mass concentration of 5 mg / mL and a dopamine-modified Ag NCs@TiO2 nanoparticle dispersion with a mass concentration of 15 mg / mL were mixed and stirred for 2 h to obtain a mixed dispersion.

[0049] The PDMS prepolymer and crosslinking agent were mixed evenly at a mass ratio of 10:1, placed in a refrigerator for 30 minutes, and the bubbles generated by stirring were removed to obtain the crosslinking solution.

[0050] The PDMS prepolymer and crosslinking agent were purchased, specifically Sylgard 184.

[0051] Wipe the surface of the glass slide (75mm×25mm) with acetone, anhydrous ethanol and water in sequence. After the surface is completely dry, 2mL of the mixed dispersion is evenly dispersed on the clean glass slide and allowed to dry naturally at room temperature for 60min. Then, 1mL of crosslinking liquid is spread on the glass slide and dried at 60℃ for 6h. After cooling, the film is peeled off from the glass slide and cut into small pieces to obtain the flexible SERS substrate material.

[0052] like Figure 3 The image shown is a SEM image of the flexible SERS substrate material. It can be seen that Ag NCs@TiO2 nanoparticles exist on the outside of the silver nanowires.

[0053] Example 2: A method for preparing a flexible SERS substrate material, specifically including the following steps:

[0054] S1. Preparation of flexible silver nanowires with high aspect ratio;

[0055] Add 0.0306 g of silver nitrate to 3 mL of deionized water, stir magnetically for 10 min until completely dissolved, store in the dark to obtain a 0.06 mol / L silver nitrate solution;

[0056] Add 0.1232 g of maltose monohydrate to 3 mL of deionized water and stir thoroughly for 10 min until completely dissolved to obtain a 0.12 mol / L reducing agent solution;

[0057] Sodium chloride was added to 2.4 mL of deionized water and stirred thoroughly for 10 min until completely dissolved, resulting in a 0.06 mol / L sodium chloride solution.

[0058] Take 27 mL of deionized water, add 1.2 g of polyvinylpyrrolidone, and stir magnetically for 30 min until completely dissolved. Then add silver nitrate solution and reducing agent solution, and continue stirring for 10 min to mix thoroughly. Then add sodium chloride solution dropwise, and continue stirring for 10 min. Then heat the reaction at 165℃ for 18 h. After cooling, a flexible silver nanowire solution is obtained. After standing and separating into layers, take the precipitate and add it to 6 mL of deionized water. Centrifuge at 3000 rpm for 5 min. Repeat three times. The precipitate is the flexible silver nanowire.

[0059] Preparation of S2, hydroxylated silver nanowire dispersion;

[0060] A 5 mg / mL hydroxylated silver nanowire dispersion was prepared by dispersing flexible silver nanowires in ethanol after alkaline heat treatment.

[0061] Specifically, the alkaline heat treatment involves adding flexible silver nanowires to a sodium hydroxide or potassium hydroxide solution, heating and stirring at 50-60°C for 10-13 hours, followed by centrifugation and washing. This method is existing technology.

[0062] Preparation of S3 and Ag NCs@TiO2 nanoparticles;

[0063] Ethylene glycol was stirred and heated at 150℃ for 1 hour. After cooling, silver nitrate, sodium sulfide hydrate, and polyvinylpyrrolidone were added to 6 mL of ethylene glycol to obtain 0.28 mol / L silver nitrate ethylene glycol solution, 3.0 mmol / L sodium sulfide ethylene glycol solution, and 0.18 mmol / L polyvinylpyrrolidone ethylene glycol solution, respectively.

[0064] Add 100 μL of sodium sulfide ethylene glycol solution to the flask, wait for 8 min, then add 1.8 mL of polyvinylpyrrolidone ethylene glycol solution, and immediately add 0.5 mL of silver nitrate ethylene glycol solution. After reacting for 10 min, centrifuge at 9000 rpm for 10 min, take the precipitate, wash it 4 times with 50% ethanol solution, and collect Ag NCs;

[0065] Under a nitrogen atmosphere, 5 μL of tetrabutyl titanate was added to 5 mL of ethanol and stirred to obtain a tetrabutyl titanate solution.

[0066] 1 mg of Ag NCs was added to 2 mL of ethanol and dispersed evenly to obtain an Ag NCs solution;

[0067] Ag NCs solution and 50 μL dimethylamine solution (40% dimethylamine aqueous solution) were mixed, sonicated and stirred for 30 min. Then tetrabutyl titanate solution was added dropwise, stirred for 1 h, heated to 165℃ and reacted for 10 h. After cooling, the precipitate was separated by centrifugation, washed three times with ethanol, and dried under vacuum at 60℃ to obtain Ag NCs@TiO2 nanoparticles.

[0068] S4. Preparation of dopamine-modified Ag NCs@TiO2 nanoparticle dispersion;

[0069] 0.1683 g Ag NCs@TiO2 nanoparticles were added to 38 mL of water and mixed thoroughly. Then, 0.2066 g of hexamethylenetetramine was added and mixed thoroughly. Finally, 0.0153 g of dopamine hydrochloride was added and the mixture was stirred at room temperature for 4 h. After centrifugation at 10000 rpm for 2 min, the precipitate was separated, washed three times with ethanol, and air-dried to obtain dopamine-modified Ag NCs@TiO2 nanoparticles.

[0070] Dopamine-modified AgNCs@TiO2 nanoparticles were dispersed in ethanol to prepare a 15 mg / mL dopamine-modified AgNCs@TiO2 nanoparticle dispersion.

[0071] S5, Preparation of flexible SERS substrate materials;

[0072] An equal volume of hydroxylated silver nanowire dispersion with a mass concentration of 5 mg / mL and a dopamine-modified Ag NCs@TiO2 nanoparticle dispersion with a mass concentration of 15 mg / mL were mixed and stirred for 2 h to obtain a mixed dispersion.

[0073] The PDMS prepolymer and crosslinking agent were mixed evenly at a mass ratio of 10:1, placed in a refrigerator for 30 minutes, and the bubbles generated by stirring were removed to obtain the crosslinking solution.

[0074] The PDMS prepolymer and crosslinking agent were purchased, specifically Sylgard 184.

[0075] Wipe the surface of the glass slide with acetone, anhydrous ethanol and water in sequence. After the surface is completely dry, 2 mL of the mixed dispersion is evenly dispersed on the clean glass slide and allowed to dry naturally at room temperature for 60 min. Then, 1 mL of crosslinking liquid is spread on the glass slide and dried at 60 °C for 6 h. After cooling, the film is peeled off the glass slide and cut into small pieces to obtain the flexible SERS substrate material.

[0076] Example 3: A method for preparing a flexible SERS substrate material, specifically including the following steps:

[0077] S1. Preparation of flexible silver nanowires with high aspect ratio;

[0078] Add 0.0306 g of silver nitrate to 3 mL of deionized water, stir magnetically for 10 min until completely dissolved, store in the dark to obtain a 0.06 mol / L silver nitrate solution;

[0079] Add 0.1232 g of maltose monohydrate to 3 mL of deionized water and stir thoroughly for 10 min until completely dissolved to obtain a 0.12 mol / L reducing agent solution;

[0080] Sodium chloride was added to 2.7 mL of deionized water and stirred thoroughly for 10 min until completely dissolved, resulting in a 0.06 mol / L sodium chloride solution.

[0081] Take 27 mL of deionized water, add 1.2 g of polyvinylpyrrolidone, and stir magnetically for 30 min until completely dissolved. Then add silver nitrate solution and reducing agent solution, and continue stirring for 10 min to mix thoroughly. Then add sodium chloride solution dropwise, and continue stirring for 10 min. Then heat the reaction at 163℃ for 19 h. After cooling, a flexible silver nanowire solution is obtained. After standing and separating into layers, take the precipitate and add it to 6 mL of deionized water. Centrifuge at 3000 rpm for 5 min. Repeat three times. The precipitate is the flexible silver nanowire.

[0082] Preparation of S2, hydroxylated silver nanowire dispersion;

[0083] A 5 mg / mL hydroxylated silver nanowire dispersion was prepared by dispersing flexible silver nanowires in ethanol after alkaline heat treatment.

[0084] Specifically, the alkaline heat treatment involves adding flexible silver nanowires to a sodium hydroxide or potassium hydroxide solution, heating and stirring at 50-60°C for 10-13 hours, followed by centrifugation and washing. This method is existing technology.

[0085] Preparation of S3 and Ag NCs@TiO2 nanoparticles;

[0086] Ethylene glycol was stirred and heated at 150℃ for 1 hour. After cooling, silver nitrate, sodium sulfide hydrate, and polyvinylpyrrolidone were added to 6 mL of ethylene glycol to obtain 0.28 mol / L silver nitrate ethylene glycol solution, 3.0 mmol / L sodium sulfide ethylene glycol solution, and 0.18 mmol / L polyvinylpyrrolidone ethylene glycol solution, respectively.

[0087] Add 100 μL of sodium sulfide ethylene glycol solution to the flask, wait for 8 min, then add 1.6 mL of polyvinylpyrrolidone ethylene glycol solution, and immediately add 0.5 mL of silver nitrate ethylene glycol solution. After reacting for 10 min, centrifuge at 9000 rpm for 10 min, take the precipitate, wash it 4 times with 50% ethanol solution, and collect Ag NCs.

[0088] Under a nitrogen atmosphere, 5 μL of tetrabutyl titanate was added to 5 mL of ethanol and stirred to obtain a tetrabutyl titanate solution.

[0089] 1 mg of Ag NCs was added to 2 mL of ethanol and dispersed evenly to obtain an Ag NCs solution;

[0090] Ag NCs solution and 50 μL dimethylamine solution (40% dimethylamine aqueous solution) were mixed, sonicated and stirred for 28 min, then tetrabutyl titanate solution was added dropwise, stirred for 1 h, heated to 163℃ and reacted for 11 h, cooled and centrifuged to separate the precipitate, washed three times with ethanol, and dried under vacuum at 60℃ to obtain Ag NCs@TiO2 nanoparticles;

[0091] S4. Preparation of dopamine-modified Ag NCs@TiO2 nanoparticle dispersion;

[0092] 0.153 g Ag NCs@TiO2 nanoparticles were added to 38 mL of water and mixed evenly. Then, 0.1989 g of hexamethylenetetramine was added and mixed evenly. Then, 0.0153 g of dopamine hydrochloride was added and the mixture was stirred at room temperature for 3 h. After centrifugation at 10000 rpm for 2 min, the precipitate was separated, washed three times with ethanol, and dried naturally to obtain dopamine-modified Ag NCs@TiO2 nanoparticles.

[0093] Dopamine-modified AgNCs@TiO2 nanoparticles were dispersed in ethanol to prepare a 15 mg / mL dopamine-modified AgNCs@TiO2 nanoparticle dispersion.

[0094] S5, Preparation of flexible SERS substrate materials;

[0095] An equal volume of hydroxylated silver nanowire dispersion with a mass concentration of 5 mg / mL and a dopamine-modified Ag NCs@TiO2 nanoparticle dispersion with a mass concentration of 15 mg / mL were mixed and stirred for 2 h to obtain a mixed dispersion.

[0096] The PDMS prepolymer and crosslinking agent were mixed evenly at a mass ratio of 10:1, placed in a refrigerator for 30 minutes, and the bubbles generated by stirring were removed to obtain the crosslinking solution.

[0097] The PDMS prepolymer and crosslinking agent were purchased, specifically Sylgard 184.

[0098] Wipe the surface of the glass slide with acetone, anhydrous ethanol and water in sequence. After the surface is completely dry, 2 mL of the mixed dispersion is evenly dispersed on the clean glass slide and allowed to dry naturally at room temperature for 60 min. Then, 1 mL of crosslinking liquid is spread on the glass slide and dried at 60 °C for 6 h. After cooling, the film is peeled off the glass slide and cut into small pieces to obtain the flexible SERS substrate material.

[0099] Example 4: Detection method for neomycin in veterinary drugs, the specific steps are as follows:

[0100] S1, Sample Pretreatment

[0101] Grind 10g of frozen pork, add 10mL of acetonitrile and 0.15mL of acetic acid, and dilute to 15mL with ultrapure water to obtain a mixed sample. Add 4g of anhydrous magnesium sulfate and 1g of sodium chloride, vortex for 3min, centrifuge at 8000r / min for 5min, take 2mL of supernatant and transfer to a purification tube containing 150mg of anhydrous magnesium sulfate and 50mg of PSA adsorbent (N-propylethylenediamine), vortex for 2min, centrifuge at 5000r / min for 3min, and take the supernatant as the sample solution to be tested.

[0102] S2. Establishment of the standard curve;

[0103] A blank sample solution was prepared from antibiotic-free blank pork according to the method in step S1;

[0104] Dissolve 1 mol of neomycin sulfate in 1 L of ultrapure water to obtain neomycin sulfate standard stock solution;

[0105] The 1 mol / L neomycin sulfate standard stock solution was serially diluted with blank sample solution to obtain a series of spiked sample solutions of different concentrations, namely 10 mol / L, ... -1 mmol / L, 10 -2 mmol / L, 10 -3 mmol / L, 10 -4 mmol / L, 10 -5 mmol / L;

[0106] 10 μL of spiked sample solution was dropped onto the surface of the flexible SERS substrate material prepared in Example 1. After drying, SERS detection was performed. The specific detection conditions were: excitation wavelength 785 nm, laser power 5 mW, and integration time 10 s.

[0107] The characteristic peak intensity of neomycin sulfate (619 cm⁻¹) -1 The standard curve is fitted with the logarithm (lgc) of the concentration of the spiked sample solution, as shown below. Figure 4As shown, the linear regression equation is obtained as: y = 1265.0lgc + 7695.0;

[0108] S3. Add 10 μL of the sample solution to the surface of the flexible SERS substrate, allow it to dry, and then perform SERS detection. Record the characteristic peak intensity (619 cm⁻¹) of the antibiotic in the SERS spectrum of the sample solution. -1 Substitute the values ​​into the linear regression equation to calculate the residual concentration of antibiotics in the sample solution.

[0109] In this embodiment, the specific method for SERS detection is existing technology, and the method for determining the characteristic peaks is also existing technology.

[0110] In this embodiment, the LOD is 5.8 × 10⁻⁶. -5 The method for determining LOD at mmol / L is existing technology.

[0111] Example 5: Detection method of apramycin in veterinary drugs. The specific steps are the same as in Example 4, except that a 1 mol / L apramycin standard stock solution is used instead of the neomycin sulfate standard stock solution.

[0112] The characteristic peak intensity of apramycin (468 cm⁻¹) -1 The standard curve is fitted with the logarithm (lgc) of the concentration of the spiked sample solution, as shown below. Figure 5 As shown, the linear regression equation is: y = 1275.0lgc + 40845.0.

[0113] In this embodiment, the LOD is 1.2 × 10⁻⁶. -5 The method for determining LOD at mmol / L is existing technology.

[0114] Comparative Example 1 This comparative example provides a method for preparing a flexible SERS substrate material. The specific steps are the same as in Example 1, except that step S5 is different. Step S5 in this comparative example is as follows:

[0115] An equal volume of hydroxylated silver nanowire dispersion with a mass concentration of 5 mg / mL and a dopamine-modified Ag NCs@TiO2 nanoparticle dispersion with a mass concentration of 15 mg / mL were mixed and stirred for 2 h to obtain a mixed dispersion.

[0116] The PDMS prepolymer and crosslinking agent were mixed evenly at a mass ratio of 10:1, placed in a refrigerator for 30 minutes, and the bubbles generated by stirring were removed to obtain the crosslinking solution.

[0117] 1 mL of crosslinking solution was spread on a glass slide and then placed in a 60°C oven to cure for 6 hours. After cooling at room temperature, a PDMS film was formed. 2 mL of mixed dispersion was drop-coated onto the PDMS film and dried at room temperature. The film was then peeled off the glass slide and cut into small pieces to obtain a flexible SERS substrate material.

[0118] Comparative Example 2 This comparative example provides a method for preparing a flexible SERS substrate material. The specific steps are the same as in Example 1, except that the flexible silver nanowires are not subjected to alkaline heat treatment and the Ag NCs@TiO2 nanoparticles are not modified with dopamine. The specific steps are as follows:

[0119] S1. Preparation of flexible silver nanowires with high aspect ratio;

[0120] Add 0.0306 g of silver nitrate to 3 mL of deionized water, stir magnetically for 10 min until completely dissolved, store in the dark to obtain a 0.06 mol / L silver nitrate solution;

[0121] Add 0.1232 g of maltose monohydrate to 3 mL of deionized water and stir thoroughly for 10 min until completely dissolved to obtain a 0.12 mol / L reducing agent solution;

[0122] Add sodium chloride to 3 mL of deionized water and stir thoroughly for 10 min until completely dissolved to obtain a 0.06 mol / L sodium chloride solution;

[0123] Take 27 mL of deionized water, add 1.2 g of polyvinylpyrrolidone, and stir magnetically for 30 min until completely dissolved. Then add silver nitrate solution and reducing agent solution, and continue stirring for 10 min to mix thoroughly. Then add sodium chloride solution dropwise, and continue stirring for 10 min. Then heat the reaction at 160℃ for 20 h. After cooling, a flexible silver nanowire solution is obtained. After standing and separating into layers, take the precipitate and add it to 6 mL of deionized water. Centrifuge at 3000 rpm for 5 min. Repeat three times. The precipitate is the flexible silver nanowire.

[0124] S2, Preparation of silver nanowire dispersion;

[0125] Flexible silver nanowires were dispersed in ethanol to prepare a 5 mg / mL silver nanowire dispersion.

[0126] Preparation of S3 and Ag NCs@TiO2 nanoparticles;

[0127] Ethylene glycol was stirred and heated at 150℃ for 1 hour. After cooling, silver nitrate, sodium sulfide hydrate, and polyvinylpyrrolidone were added to 6 mL of ethylene glycol to obtain 0.28 mol / L silver nitrate ethylene glycol solution, 3.0 mmol / L sodium sulfide ethylene glycol solution, and 0.18 mmol / L polyvinylpyrrolidone ethylene glycol solution, respectively.

[0128] Add 100 μL of sodium sulfide ethylene glycol solution to the flask, wait for 8 min, then add 1.5 mL of polyvinylpyrrolidone ethylene glycol solution, and immediately add 0.5 mL of silver nitrate ethylene glycol solution. After reacting for 10 min, centrifuge at 9000 rpm for 10 min, take the precipitate, wash it 4 times with 50% ethanol solution, and collect Ag NCs;

[0129] Under a nitrogen atmosphere, 5 μL of tetrabutyl titanate was added to 5 mL of ethanol and stirred to obtain a tetrabutyl titanate solution.

[0130] 1 mg of Ag NCs was added to 2 mL of ethanol and dispersed evenly to obtain an Ag NCs solution;

[0131] Ag NCs solution and 50 μL dimethylamine solution (40% dimethylamine aqueous solution) were mixed, sonicated and stirred for 25 min, then tetrabutyl titanate solution was added dropwise, stirred for 1 h, heated to 160℃ and reacted for 12 h, cooled and centrifuged to separate the precipitate, washed three times with ethanol, and dried under vacuum at 60℃ to obtain Ag NCs@TiO2 nanoparticles;

[0132] Preparation of S4, Ag NCs@TiO2 nanoparticle dispersion;

[0133] Ag NCs@TiO2 nanoparticles were dispersed in ethanol to prepare a 15 mg / mL Ag NCs@TiO2 nanoparticle dispersion.

[0134] S5, Preparation of flexible SERS substrate materials;

[0135] An equal volume of silver nanowire dispersion with a mass concentration of 5 mg / mL and Ag NCs@TiO2 nanoparticle dispersion with a mass concentration of 15 mg / mL were mixed and stirred for 2 h to obtain a mixed dispersion.

[0136] The PDMS prepolymer and crosslinking agent were mixed evenly at a mass ratio of 10:1, placed in a refrigerator for 30 minutes, and the bubbles generated by stirring were removed to obtain the crosslinking solution.

[0137] The PDMS prepolymer and crosslinking agent were purchased, specifically Sylgard 184.

[0138] Wipe the surface of the glass slide with acetone, anhydrous ethanol and water in sequence. After the surface is completely dry, 2 mL of the mixed dispersion is evenly dispersed on the clean glass slide and allowed to dry naturally at room temperature for 60 min. Then, 1 mL of crosslinking liquid is spread on the glass slide and dried at 60 °C for 6 h. After cooling, the film is peeled off the glass slide and cut into small pieces to obtain the flexible SERS substrate material.

[0139] Comparative Example 3 This comparative example provides a method for preparing a flexible SERS substrate material. The specific steps are the same as in Example 1, except that Ag nanospheres (Ag NSs) are used instead of Ag NCs. The preparation method of Ag NSs is as follows:

[0140] Prepare a 40 mL aqueous solution of ascorbic acid and sodium citrate, wherein the concentration of ascorbic acid is 0.6 mmol / L and the concentration of sodium citrate is 3.0 mmol / L;

[0141] The pH of a mixed aqueous solution of ascorbic acid and sodium citrate was adjusted to 10.5 using 0.1 mol / L sodium hydroxide solution. Then, 0.4 mL of 0.1 mol / L silver nitrate solution was added to the solution at 30 °C and 400 rpm until the solution turned brown. After 15 min, no further changes occurred. The solution was then heated at 80 °C for 2 h. After cooling to room temperature, the precipitate was separated by centrifugation, which was Ag NSs.

[0142] Comparative Example 4 This comparative example provides a method for preparing a flexible SERS substrate material. The specific steps are the same as in Example 1, except that in step S5, only a hydroxylated silver nanowire dispersion is added. Step S5 specifically involves:

[0143] The PDMS prepolymer and crosslinking agent were mixed evenly at a mass ratio of 10:1, placed in a refrigerator for 30 minutes, and the bubbles generated by stirring were removed to obtain the crosslinking solution.

[0144] The PDMS prepolymer and crosslinking agent were purchased, specifically Sylgard 184.

[0145] The surface of the glass slide was wiped with acetone, anhydrous ethanol and water in sequence. After the surface was completely dry, 2 mL of hydroxylated silver nanowire dispersion was evenly dispersed on the clean glass slide and allowed to dry naturally at room temperature for 60 min. Then, 1 mL of crosslinking liquid was spread on the glass slide and dried at 60 °C for 6 h. After cooling, the film was peeled off the glass slide and cut into small pieces to obtain the flexible SERS substrate material.

[0146] The sensitivity of the flexible SERS substrate materials prepared in Example 1 and Comparative Examples 1-4 was compared by performing SERS tests. The specific method is as follows:

[0147] A blank sample solution was prepared from antibiotic-free blank pork according to the method in step S1 of Example 4;

[0148] Dissolve 1 mol of apramycin in 1 L of ultrapure water to obtain apramycin standard stock solution;

[0149] Dilute the 1 mol / L apramycin standard stock solution with blank sample solution to obtain a concentration of 10.-2 Spiked sample solutions at mmol / L;

[0150] 10 μL of spiked sample solution was added dropwise to the surface of the flexible SERS substrate materials prepared in Example 1 and Comparative Examples 1-4, respectively. After drying, SERS detection was performed. The specific detection conditions were: excitation wavelength 785 nm, laser power 5 mW, and integration time 10 s. The characteristic peak intensity (468 cm⁻¹) of each group of apramycin was obtained. -1 The peak intensity of each substrate material was measured at 5 points in parallel and the average value was taken, as shown in Table 1.

[0151] Table 1. Peak intensity results of flexible SERS substrate materials in Example 1 and Comparative Examples 1-4

[0152]

[0153] As can be seen from Table 1, the peak intensity of the flexible SERS substrate material in Example 1 is the highest, indicating that the flexible SERS substrate material prepared by the present invention has the best surface reinforcement effect and the highest sensitivity.

[0154] Compared with Example 1, the peak intensity of the detection results of Comparative Example 1 is similar, indicating that there is little difference in the SERS detection sensitivity between embedded substrate materials and adhesive substrate materials.

[0155] Compared with Example 1, the peak intensity of the detection results of Comparative Example 2 decreased significantly. This is because the silver nanowires and Ag NCs@TiO2 nanoparticles in Comparative Example 2 are randomly distributed in the base material and each plays an individual role in enhancing the effect. Therefore, the enhancement effect is significantly lower than that of Example 1, and the sensitivity is relatively low.

[0156] Compared with Example 1, the peak intensity of the detection results of Comparative Example 3 also decreased to a certain extent. This is because Ag NSs was used in Comparative Example 3, and its Raman enhancement effect was lower than that of Ag NCs.

[0157] Compared with Example 1, the peak intensity of Comparative Example 4 also decreased significantly. This is because no Ag NCs@TiO2 nanoparticles were added in Comparative Example 4, resulting in a low Raman enhancement effect.

[0158] The reusability of the flexible SERS substrate materials of Example 1 and Comparative Example 1 was determined by performing five consecutive cycles of SERS testing on the flexible SERS substrate materials.

[0159] A blank sample solution was prepared from antibiotic-free blank pork according to the method in step S1 of Example 4;

[0160] Dissolve 1 mol of apramycin in 1 L of ultrapure water to obtain apramycin standard stock solution;

[0161] Dilute the 1 mol / L apramycin standard stock solution with blank sample solution to obtain a concentration of 10. -2 Spiked sample solutions at mmol / L;

[0162] 10 μL of spiked sample solution was added dropwise to the flexible SERS substrates of Example 1 and Comparative Example 1, respectively. After drying, SERS detection was performed. After washing with ethanol and water 5 times, spiked sample solution was added again and SERS detection was repeated 5 times. The characteristic peak intensity (468 cm⁻¹) of apramycin was obtained for each cycle. -1 In each cycle, the peak intensity of 5 points on the flexible SERS substrate was measured in parallel and the average value was taken. The results are shown in Table 2.

[0163] Table 2. Test results of the reusability of the substrate materials in Example 1 and Comparative Example 1

[0164]

[0165] As can be seen from Table 2, the test results of the flexible SERS substrate material in Example 1 remained basically unchanged after being repeatedly used 5 times, indicating that its repeated use effect was good. However, the peak intensity of the SERS substrate material in Comparative Example 1 decreased to a certain extent during repeated use.

[0166] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting neomycin and apramycin in veterinary drugs, characterized in that, Includes the following steps: S1. Sample pretreatment; Grind the frozen sample, add acetonitrile and acetic acid dropwise, and make up to 15 mL to obtain a mixed sample. Add anhydrous magnesium sulfate and sodium chloride to the mixed sample, vortex and centrifuge, and transfer the supernatant to a mixture of anhydrous magnesium sulfate and PSA adsorbent. Vortex and centrifuge, and take the supernatant as the sample solution to be tested. S2. Establishment of the standard curve; A blank sample solution was prepared from a blank sample that does not contain antibiotics according to the method in step S1; The 1 mol / L antibiotic standard stock solution was serially diluted with blank sample solution to obtain a series of spiked sample solutions. The spiked sample solution was dropped onto the surface of a flexible SERS substrate material, dried, and then subjected to SERS detection. A linear regression equation was fitted using the logarithm of the characteristic peak intensity of the antibiotic and the concentration of the spiked sample solution. S3. Drop the sample solution to be tested onto the surface of the flexible SERS substrate material, let it dry, and then perform SERS detection. Record the characteristic peak intensity of antibiotics in the SERS spectrum of the sample solution to be tested, substitute it into the linear regression equation, and calculate the residual concentration of antibiotics in the sample solution to be tested. The method for preparing the flexible SERS substrate material is as follows: A mixed dispersion was obtained by mixing equal volumes of hydroxylated silver nanowire dispersion and dopamine-modified Ag NCs@TiO2 nanoparticle dispersion. The PDMS prepolymer and crosslinking agent were mixed evenly, and the mixture was placed in a refrigerator to remove air bubbles, thus obtaining a crosslinking solution. Wipe the surface of the glass slide and wait for it to dry completely. Then, disperse the mixed dispersion on the glass slide and let it dry naturally at room temperature. Next, spread the crosslinking liquid on the glass slide and dry it. Finally, peel the film off the glass slide and cut it into small pieces to obtain the flexible SERS substrate material.

2. The method for detecting neomycin and apramycin in veterinary drugs according to claim 1, characterized in that, The preparation method of the hydroxylated silver nanowire dispersion is as follows: Maltose monohydrate was added to deionized water and stirred thoroughly until completely dissolved to prepare a 0.12 mol / L reducing agent solution. Polyvinylpyrrolidone was dissolved in deionized water, and 0.06 mol / L silver nitrate solution and reducing agent solution were added. After stirring and mixing thoroughly, 0.06 mol / L sodium chloride solution was added dropwise, and stirring and mixing continued. The mixture was then heated to 160-165℃ and reacted for 18-20 hours. After cooling, the mixture was allowed to stand and separate into layers. The precipitate was washed three times with deionized water to obtain flexible silver nanowires. The flexible silver nanowires were then subjected to alkaline heat treatment and dispersed in ethanol to obtain a hydroxylated silver nanowire dispersion.

3. The method for detecting neomycin and apramycin in veterinary drugs according to claim 2, characterized in that, The volume ratio of the silver nitrate solution, reducing agent solution, and sodium chloride solution is 1:1:0.8~1, and the mass-volume ratio of the polyvinylpyrrolidone and silver nitrate solution is 4g:1mL.

4. The method for detecting neomycin and apramycin in veterinary drugs according to claim 2, characterized in that, The preparation method of dopamine-modified Ag NCs@TiO2 nanoparticle dispersion is as follows: Preparation of Ag NCs@TiO2 nanoparticles; Tetrabutyl titanate was added to ethanol under a nitrogen atmosphere and stirred to obtain a tetrabutyl titanate solution. Ag NCs were added to ethanol and dispersed evenly to obtain an Ag NCs solution; AgNCs solution and dimethylamine solution were mixed, sonicated and stirred for 25-30 min, then tetrabutyl titanate solution was added dropwise, stirred for 1 h, heated to 160-165℃ and reacted for 10-12 h, cooled and centrifuged to separate the precipitate, washed and dried to obtain AgNCs@TiO2 nanoparticles. Preparation of dopamine-modified Ag NCs@TiO2 nanoparticles; Ag NCs@TiO2 nanoparticles were added to water and mixed evenly. Then, hexamethylenetetramine was added and mixed evenly. Dopamine hydrochloride was added and the mixture was stirred at room temperature for 2-4 hours. After centrifugation, the precipitate was separated, washed, dried, and then redispersed in ethanol to obtain dopamine-modified Ag NCs@TiO2 nanoparticles.

5. The method for detecting neomycin and aspirin in veterinary drugs according to claim 4, characterized in that, The preparation method of AgNCs is as follows: 0.18 mmol / L polyvinylpyrrolidone glycol solution is added to 3.0 mmol / L sodium sulfide glycol solution, and then 0.28 mol / L silver nitrate glycol solution is immediately added. After reacting for 10 min, the mixture is centrifuged, the precipitate is separated, and after washing, AgNCs are obtained.

6. The method for detecting neomycin and apramycin in veterinary drugs according to claim 5, characterized in that, The volume ratio of the polyvinylpyrrolidone ethylene glycol solution, sodium sulfide ethylene glycol solution, and silver nitrate ethylene glycol solution is 15~18:1:

5.

7. The method for detecting neomycin and apramycin in veterinary drugs according to claim 4, characterized in that, In the preparation of Ag NCs@TiO2 nanoparticles, the volume ratio of dimethylamine solution to tetrabutyl titanate is 10:1, and the mass-volume ratio of Ag NCs to tetrabutyl titanate is 1 mg: 5 μL. In the preparation of dopamine-modified Ag NCs@TiO2 nanoparticles, the mass ratio of Ag NCs@TiO2 nanoparticles, hexamethylenetetramine, and dopamine hydrochloride is 10~11:13~13.5:

1.

8. The method for detecting neomycin and apramycin in veterinary drugs according to claim 1, characterized in that, In step S1, the volume fraction of acetic acid in the mixed sample is 0.5-1%, and the mass of the frozen sample is 10-12g.

9. The method for detecting neomycin and apramycin in veterinary drugs according to claim 1, characterized in that, In the preparation method of the flexible SERS substrate material, the mass concentrations of the hydroxylated silver nanowire dispersion and the dopamine-modified Ag NCs@TiO2 nanoparticle dispersion are 5 mg / mL and 15 mg / mL, respectively; the mass ratio of the PDMS prepolymer to the crosslinking agent is 10:1; and the volume ratio of the mixed dispersion to the crosslinking liquid is 2:

1.

10. The method for detecting neomycin and apramycin in veterinary drugs according to claim 1, characterized in that, The antibiotic is neomycin sulfate or apramycin.

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