A method for detecting malachite green veterinary drug residues based on SERS using gold-plated nanorods

Abstract

This invention provides a method for detecting malachite green veterinary drug residues using SERS based on silver-coated gold nanorods. The method includes: mixing a malachite green standard solution with silver-coated gold nanorods, adding a coagulant to form a mixed solution, and performing Raman spectroscopy to obtain the surface-enhanced Raman scattering (SERS) peaks of the malachite green standard solution; determining the characteristic peaks used to identify malachite green using SERS by assigning the malachite green molecular structure to the Raman peak positions; and determining the linear relationship between the concentration of malachite green and the intensity of the characteristic peaks. The method also involves mixing the sample solution to be tested with silver-coated gold nanorods, adding a coagulant to form a mixed solution, and performing Raman spectroscopy to obtain the peak intensity of the characteristic peaks of malachite green in the sample solution. The concentration of malachite green in the sample solution is calculated based on the linear relationship between the concentration of malachite green and the peak intensity of the characteristic peaks. This invention enables accurate and rapid detection of malachite green in aquatic products, ensuring the safe production of aquatic products.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection technology, specifically to a method for detecting malachite green veterinary drug residues based on SERS using silver-coated gold nanorods. Background Technology

[0002] Veterinary drug residues are among the most common hazards to food safety, posing serious risks to human health. Long-term consumption of agricultural products with excessive levels of veterinary drugs can cause "three-fold" effects (carcinogenic, teratogenic, and mutagenic), leading to chronic poisoning, allergic reactions in some individuals, and precocious puberty in adolescents and children. Simultaneously, it severely impacts the market sales and export of agricultural products. Malachite green, for example, is a toxic triphenylmethane-based basic dye that can be used as a dye and as a chemical agent for sterilization and antiparasitic purposes. Malachite green can control and treat parasitic, fungal, and microbial infections in aquatic products. Studies have shown that malachite green has a long degradation time and exhibits severe teratogenic, mutagenic, and carcinogenic effects on humans. In many countries, including China, the United States, and Japan, its use as a bactericide in aquaculture is prohibited. China's Ministry of Agriculture and Rural Affairs has included malachite green in its list of prohibited drugs and other compounds for use in food animals. In aquaculture, malachite green continues to be illegally used due to its low cost and high effectiveness against fungal and parasitic infections, significantly impacting consumer health and export trade. Currently, traditional analytical techniques for malachite green detection include liquid chromatography / mass spectrometry (LC / MS), high-performance liquid chromatography (HPLC), spectrophotometry, and biosensors. However, these methods have drawbacks, such as expensive equipment, cumbersome operation, complex pretreatment, and long response times. Therefore, developing rapid and accurate analytical methods is crucial for monitoring both the aquaculture environment and the aquatic products themselves.

[0003] Surface-enhanced Raman scattering (SERS) spectroscopy, due to its high sensitivity, high resolution, and ability to provide rich structural information, enables qualitative and quantitative detection and is widely used in food monitoring and trace analysis of samples. SERS detection methods are simple, rapid, and easy to operate, especially portable Raman rapid detectors, which have demonstrated considerable advantages in food safety monitoring. However, their practical application is currently quite limited, mainly because most target analytes produce weak Raman signals, Raman enhancers have limited effectiveness, and matrix interference significantly affects detection sensitivity and accuracy.

[0004] A search revealed that Chinese patent application publication number CN108672716A discloses a method for preparing silver-coated gold nanorods. However, the above patent has the following shortcomings: although the above method can prepare silver-coated gold nanorods and has a certain SERS enhancement effect, its stability and enhancement effect as a SERS enhancer are poor, which greatly limits its application in the field of SERS detection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for detecting malachite green residues based on SERS using silver-coated gold nanorods.

[0006] This invention provides a method for detecting malachite green residues based on SERS using silver-coated gold nanorods, comprising:

[0007] Silver-coated gold nanorods were provided as Raman scattering enhancers and as a standard solution of malachite green, the analyte.

[0008] The malachite green standard solution was mixed with the silver-coated gold nanorods, and a coagulating agent was added to form a first mixed solution. The first mixed solution was subjected to Raman spectroscopy to obtain the surface-enhanced Raman scattering (SERS) peaks of the malachite green standard solution. The characteristic peaks used to identify malachite green by SERS were determined by using the molecular structure of malachite green and the Raman peak positions. The linear relationship between the concentration of malachite green and the peak intensity of the characteristic peaks was also determined.

[0009] The sample solution to be tested is mixed with the silver-coated gold nanorods, and then a coagulant is added to form a second mixed solution. The second mixed solution is subjected to Raman spectroscopy to obtain the peak intensity of the characteristic peak of malachite green in the sample solution to be tested. The concentration of malachite green in the sample solution to be tested is calculated by the linear relationship between the concentration of malachite green and the peak intensity of the characteristic peak, that is, the residual amount of malachite green is obtained.

[0010] Preferably, the mass percentages of the sample solution to be tested, the silver-coated gold nanorods, and the coagulant are 10:10:3.

[0011] Preferably, the coagulant is any one of NaCl, KI, Na2SO4, and NaBr.

[0012] Preferably, the method for detecting malachite green residue based on SERS of silver-coated gold nanorods further includes preparing the silver-coated gold nanorods:

[0013] S1, Synthesis of gold seeds: Mix 0.1 mL to 1.0 mL of 0.01 mol / L HAuCl4 solution with 1 mL to 12 mL of 0.1 mol / L hexadecyltrimethylammonium bromide solution to obtain a first solution. Then, quickly add 0.1 mL to 0.8 mL of 0.01 mol / L NaBH4 solution in an ice bath to the first solution to form a first reaction solution. After incubating the first reaction solution at a constant temperature, a brown gold seed solution is obtained.

[0014] S2, Synthesis of Gold Nanorods: 1-4 mL of 0.01 mol / L AgNO3 was mixed thoroughly with a binary surfactant solution consisting of 10-80 mL of 0.08 M hexadecyltrimethylammonium bromide and 0.02 M sodium oleate to form a second solution. This second solution was incubated at a constant temperature. Then, 10-40 mL of 0.001 mol / L HAuCl4 solution was added to the incubated second solution. After stirring at 500-1000 rpm for 20-100 minutes, the solution color changed from bright yellow to colorless. 1-5 mL of 1 mol / L HCl was added, and the mixture was stirred for 5-20 minutes. Finally, 20-200 μL of [a specific solution] was added under vigorous stirring. A growth solution was prepared by mixing 0.1 mol / L ascorbic acid solution with gold seed solution. 20 μL-100 μL of the gold seed solution was added to the growth solution while stirring at 300 rpm-800 rpm to obtain a second reaction solution. The second reaction solution was incubated at a constant temperature and then centrifuged to obtain the gold nanorods.

[0015] S3, Synthesis of silver-coated gold nanorods: 50 μL-400 μL of 0.01 mol / L AgNO3 and 200 μL-800 μL of 0.08 M hexadecyltrimethylammonium chloride are mixed evenly. After constant temperature incubation, 2 mL-4 mL of the gold nanorods prepared in step S2 are added. After constant temperature incubation, 20 μL-200 μL of 0.1 mol / L ascorbic acid solution is added under vigorous stirring to obtain a mixed solution. After constant temperature incubation and centrifugation, the silver-coated gold nanorods are obtained. The silver-coated gold nanorods are then uniformly dispersed in an aqueous phase and inert gas is introduced to fully remove dissolved oxygen in the solution, prevent the silver shell from being oxidized, and improve the SERS stability of the silver-coated gold nanorods.

[0016] Preferably, the method for detecting malachite green veterinary drug residues based on SERS using silver-coated gold nanorods further includes: pretreating the sample to prepare the test sample solution.

[0017] Place the sample and hydroxylamine hydrochloride in a centrifuge tube, let stand for 5-12 minutes, then add acetonitrile and anhydrous magnesium sulfate for vortex extraction; then add neutral alumina for vortex extraction, centrifuge at 4000-10000 rpm / min for 4-10 minutes, take out the supernatant and dry it with nitrogen to obtain the dried product; add 2,3-dichloro-5,6-dicyano-1,4-benzoquinone acetonitrile solution to the dried product for redissolution, shake for 2-12 minutes, then add neutral alumina for vortex extraction, centrifuge for 4-12 minutes, and filter to obtain the sample solution to be tested.

[0018] Preferably, under the conditions of a wavelength of 785 nm, a laser power of 10%-100%, an integration time of 1 s-20 s, and 2-4 integration times, the surface-enhanced Raman scattering peak of the malachite green standard solution appears at 1167 cm⁻¹. -1 1361cm -1 1390cm -1 1612cm -1 Place.

[0019] Preferably, the characteristic peak for determining malachite green in surface-enhanced Raman scattering spectroscopy is located at 1167 cm⁻¹. -1 Place.

[0020] Preferably, the method is used to detect malachite green residues in aquatic products, and the detection limit for malachite green residues in aquatic products is 1.58 × 10⁻⁶. -9 M.

[0021] Preferably, the concentration of the malachite green is measured within a range of 5 × 10⁻⁶. -9 M-2×10 -7 M.

[0022] Preferably, the amount of silver-coated gold nanorods added is 50 μL-400 μL.

[0023] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0024] The method described in this invention utilizes silver-coated gold nanorods as a Raman scattering enhancer, precisely controls the thickness of the silver shell, and protects the silver-coated gold nanorod material by filling it with inert gas or nitrogen to improve the stability of the SERS enhancer. Furthermore, a coagulant is added to the silver-coated gold nanorods to enhance the SERS "hot spots," thereby significantly enhancing the Raman signal of molecules. This enables accurate and rapid detection of malachite green content in aquatic products, allowing for real-time monitoring of malachite green content in aquatic products and ensuring safe production.

[0025] The method described above in this invention has a measurement range of 5 × 10⁻⁶ for malachite green.-9 M-2×10 -7 M, the limit of detection (LOD) is 1.58 × 10⁻⁶. -9 M.

[0026] The method described above in this invention provides a simple, low-cost, and easily commercialized silver-coated gold nanorod preparation process suitable for large-scale production. Attached Figure Description

[0027] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0028] Figure 1 This is a schematic diagram illustrating the principle of a preferred embodiment of the method for detecting malachite green veterinary drug residues based on SERS using silver-coated gold nanorods according to the present invention.

[0029] Figure 2 This is a schematic diagram illustrating the principle of the method for detecting malachite green veterinary drug residues based on SERS using silver-coated gold nanorods in Example 1.

[0030] Figure 3 This is a transmission electron microscope image of the silver-coated gold nanorods in Example 1;

[0031] Figure 4 The image shows the ultraviolet spectrum of the silver-coated gold nanorods in Example 1.

[0032] Figure 5 The SERS spectra of different concentrations of malachite green in Example 1 are shown below (a: 2×10⁻⁷ M; b: 10⁻⁸ M; c: 5×10⁻⁸ M; d: 2×10⁻⁸ M; e: 10⁻⁸ M; f: 5×10⁻⁹ M).

[0033] Figure 6 This represents the linear relationship between different concentrations of malachite green and the intensity of the characteristic peak in Example 1. Detailed Implementation

[0034] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0035] This invention creatively controls the dosage of AgNO3, optimizes the type and concentration of coagulants, and further introduces inert gas as a protective agent to prepare silver-coated gold nanorods with different silver shell thicknesses. It also screens out silver-coated gold nanorods with the optimal core-shell thickness for SERS enhancement, thereby improving the stability of SERS enhancers and providing a reliable SERS enhancer for practical application in the field of SERS detection.

[0036] A preferred embodiment of the present invention provides a method for detecting malachite green veterinary drug residues based on SERS using silver-coated gold nanorods, referring to... Figure 1 As shown, it includes the following steps:

[0037] Silver-coated gold nanorods were provided as Raman scattering enhancers and as a standard solution of malachite green, the analyte.

[0038] Malachite green standard solution was mixed with silver-coated gold nanorods, and a coagulant was added to form a first mixed solution. Raman spectroscopy was performed on the first mixed solution to obtain the surface-enhanced Raman scattering (SERS) peaks of the malachite green standard solution. By using the molecular structure of malachite green and the Raman peak position assignment, the characteristic peaks used to identify malachite green in SERS were determined, and the linear relationship between the concentration of malachite green and the peak intensity of the characteristic peaks was determined.

[0039] The sample solution to be tested was mixed with silver-coated gold nanorods, and then a coagulant was added to form a second mixed solution. Raman spectroscopy was performed on the second mixed solution to obtain the peak intensity of the characteristic peak of malachite green in the sample solution. The residual amount of malachite green was obtained by observing the linear relationship between the concentration of malachite green and the peak intensity of the characteristic peak. In one embodiment, NaCl can be used as a coagulant, which is beneficial for highly selectively enhancing the SERS signal of malachite green. In another embodiment, the mass percentages of the sample solution to be tested, silver-coated gold nanorods, and coagulant are 10:5:2. In other embodiments, the concentration of the malachite green standard solution is 5 × 10⁻⁶. -9 M-2×10 -7 M, the amount of silver-coated gold nanorods added can be 50-400 μL.

[0040] In specific implementation, Ag is used. + Gold nanorods were prepared using an assisted seed growth method. By controlling the amount of AgNO3 added, a silver-coated gold nanorod Raman scattering enhancer was prepared, which is beneficial to improving the stability of the silver-coated gold nanorod Raman scattering enhancer.

[0041] In other preferred embodiments, Raman spectroscopy detection was performed at a wavelength of 785 nm, a laser power of 10%-100%, an integration time of 1 s-20 s, and 2-4 integration times. The surface-enhanced Raman scattering peak of the malachite green standard solution appeared at 1167 cm⁻¹. -11361cm -1 1390cm -1 1612cm -1 Location. In one embodiment, 1167 cm was determined. -1 The characteristic peak at 1167 cm⁻¹ was used as the discrimination criterion for the detection of malachite green by surface-enhanced Raman scattering spectroscopy, and the relationship between malachite green concentration and 1167 cm⁻¹ was determined. -1 The linear relationship between peak intensity and peak intensity.

[0042] In other preferred embodiments, the method for detecting malachite green residue based on SERS of silver-coated gold nanorods further includes preparing silver-coated gold nanorods, as described above. Figure 2 As shown, the following steps are performed:

[0043] S1, Synthesis of gold seeds: Mix 0.1 mL to 1.0 mL of 0.01 mol / L HAuCl4 solution with 1 mL to 12 mL of 0.1 mol / L hexadecyltrimethylammonium bromide (CTAB) solution and stir until homogeneous to obtain the first solution. Then, quickly add 0.1 mL to 0.8 mL of 0.01 mol / L NaBH4 solution (in an ice bath) to the first solution to form the first reaction solution. Stir for 1 to 5 minutes. Place the first reaction solution in a 30-40℃ forced-air drying oven for constant temperature incubation for 1 to 5 hours to obtain a brown gold seed solution.

[0044] S2, Synthesis of Gold Nanorods: Mix 1-4 mL of 0.01 mol / L AgNO3 with a binary surfactant solution consisting of 10-80 mL of 0.08 M hexadecyltrimethylammonium bromide (CTAB) and 0.02 M sodium oleate (NaOL) to form a second solution. Stir at 300-1000 rpm for 1-10 min, and incubate at 37℃ for 15 min. Then add 10-40 mL of 0.001 mol / L HAuCl4 solution to the second solution after incubation. Stir at 500-1000 rpm for 20-100 min until the solution changes from bright yellow to colorless. Add 1-5 mL of 1 mol / L... After stirring with HCl at 200-800 rpm for 5-20 minutes, 20-200 μL of 0.1 mol / L ascorbic acid solution (L-AA) was added under vigorous stirring to obtain a growth solution. Then, 20-100 μL of gold seed solution was added to the growth solution under stirring at 300-800 rpm for 1-10 minutes to obtain a second reaction solution. This second reaction solution was incubated at 20-40℃ for 2-20 hours, and then centrifuged to obtain gold nanorods. In one embodiment, the centrifugation process can be performed as follows: the gold nanorods obtained after incubation are centrifuged twice at high speed, the supernatant is removed, and ultrapure water is added to a final volume of 30 mL. After vortexing, the mixture is stored for later use.

[0045] S3, Synthesis of Silver-Coated Gold Nanorods: 50 μL-400 μL of 0.01 mol / L AgNO3 and 200-800 μL of 0.08 M hexadecyltrimethylammonium chloride (CTAC) are mixed thoroughly and incubated in an oven at 20-40℃ for 1-20 min. Then, 2 mL-4 mL of the gold nanorods prepared in step S2 are added, and the mixture is incubated at 20-40℃ for another 1-10 min. Then, 20 μL-200 μL of 0.1 mol / L ascorbic acid solution (L-AA) is added under vigorous stirring. The resulting mixture is incubated at 20-40℃ for 1-10 h, and centrifuged to obtain the silver-coated gold nanorods. In one embodiment, the centrifugation process can be performed as follows: the prepared silver-coated gold nanorods are centrifuged twice at 4000-10000 rpm, brought to the original volume, an inert gas protective agent is added, the mixture is vortexed, and then stored for later use.

[0046] In other preferred embodiments, the method for detecting malachite green residues based on SERS using silver-coated gold nanorods further includes: pretreating aquatic product samples to prepare a sample solution for testing.

[0047] Place 2.00g of aquatic product sample and 0.2-2.0ml of 6-20g / L hydroxylamine hydrochloride in a 50ml capped plastic centrifuge tube. Let stand for 2-30min, then add 10-50ml of acetonitrile and 0.2-2.0mol / L anhydrous magnesium sulfate for vortex extraction. Add 1-10g of neutral alumina for vortex extraction, centrifuge at 4000-10000rpm / min for 2-6min, and remove the supernatant for nitrogen blowing to obtain the dried product. Add 1-5ml of 0.1-2mmol / L 2,3-dichloro-5,6-dicyano-1,4-benzoquinone acetonitrile solution to the dried product for redissolution, shake for 2-12min, then add 0.1-2g of neutral alumina for vortex extraction for 10-60s, centrifuge at 12000rpm / min for 8-12min, and filter using a 0.22μm PTFE needle filter membrane to obtain the sample solution to be tested.

[0048] In other preferred embodiments, the method for detecting malachite green residues based on SERS using silver-coated gold nanorods is used to detect malachite green residues in aquatic products, with a detection limit of 1.58 × 10⁻⁶ for malachite green residues in aquatic products. - 9 M. The concentration range for malachite green is 5 × 10⁻⁶. -9 M-2×10 -7 M.

[0049] The following description is based on specific embodiments.

[0050] Example 1

[0051] A method for detecting malachite green residues in Procambarus clarkii based on SERS using silver-coated gold nanorods is provided, comprising the following steps:

[0052] The silver-coated gold nanorods were prepared according to the following steps:

[0053] S11, Synthesis of gold seeds: Add 0.20 mL of 0.01 mol / L HAuCl4 solution to 10 mL of 0.1 mol / L hexadecyltrimethylammonium bromide (CTAB) solution, stir well, quickly add 0.5 mL of 0.01 mol / L NaBH4 solution in an ice bath, stir for 5 min, and then place the reaction solution in a 37℃ forced-air drying oven for constant temperature incubation for 3 h to obtain brown seed growth solution.

[0054] S12, Synthesis of Gold Nanorods: 1 mL of 0.01 mol / L AgNO3 was added to 50 mL of a binary surfactant solution composed of 0.1 M CTAB and 0.01 M sodium oleate (NaOL). The mixture was stirred at 500 rpm for 5 min, and then incubated at 37 °C for 20 min. Then, 10 mL of 0.001 mol / L HAuCl4 solution was added, and the mixture was stirred at 500 rpm for 90 min. The solution color changed from bright yellow to colorless. 2 mL of 2 mol / L HCl was added, and the mixture was stirred at 500 rpm for 20 min. Then, 50 μL of 0.1 mol / L ascorbic acid (L-AA) solution was added under vigorous stirring to obtain the growth solution. Finally, 20 μL of gold seed solution was added at 500 rpm and stirred for 2 min. The mixture was incubated at 37 °C for 20 h. The prepared gold nanorods were centrifuged twice at high speed, the supernatant was removed, and ultrapure water was added to a final volume of 30 mL. After vortexing, the mixture was stored for later use.

[0055] S13, Synthesis of Silver-Coated Gold Nanorods: 100 μL of 0.01 mol / L AgNO3 was mixed with 500 μL of 0.08 MCTAC and incubated at 37 °C for 20 min. Then, 3 mL of the gold nanorods prepared in step S12 was added, and the mixture was incubated at 37 °C for 10 min. Then, 50 μL of 0.1 mol / L L-AA solution was added under vigorous stirring. The resulting mixture was incubated at 37 °C for 4 h to obtain silver-coated gold nanorods. (Refer to...) Figure 3 , Figure 4 As shown. The prepared silver-coated gold nanorods were centrifuged twice at 6000 rpm, brought to the original volume, and then filled with inert argon gas as a protective agent. After vortex mixing, they were stored for later use.

[0056] SERS detection of malachite green standard solution:

[0057] Take 10g of malachite green standard solution -10-10 -6 100 μL of M was vortexed with 100 μL of silver-coated gold nanorods, and 50 μL of 0.1 M NaCl was added and vortexed again. 10 μL of the mixture was transferred onto a glass slide using a pipette to form spherical droplets. The slide was allowed to air dry for 10 min, and the area to be detected was then subjected to Raman spectroscopy using a portable Raman spectrometer. The test conditions were: detection wavelength of 785 nm, laser power of 80%, integration time of 10 s, and integration count of 3.

[0058] SERS spectral determination of malachite green and SERS analysis of standards:

[0059] Reference Figure 5 As shown, a 793 cm⁻¹ was observed in the SERS spectrum of malachite green. -1 1167cm -1 1213cm -1 1293cm -1 1363cm -1 1391cm -1 1612cm -1 Based on the molecular structure of malachite green and the Raman peak assignment, the characteristic SERS peak at 1167 cm⁻¹ was determined. -1 The characteristic peaks at these locations are used as the criteria for detecting malachite green using SERS spectroscopy. The intensity of the characteristic peaks in the SERS spectrum of malachite green varies with the concentration of the standard solution, as referenced... Figure 6 As shown, the concentration of malachite green is related to 1167 cm⁻¹. -1 The linear regression equation for the characteristic peak intensity at a given location is: y = 19863.15logx + 176665.03, with a correlation coefficient R0. 2 =0.989.

[0060] Weigh 2.00 g of homogenized sample of Procambarus clarkii, 0.2 ml of 9.5 g / L hydroxylamine hydrochloride, and place them in a 50 ml capped plastic centrifuge tube. After standing for 10 min, add 5 ml of acetonitrile and 2.0 mol / L anhydrous magnesium sulfate for vortex extraction. Add 2 g of neutral alumina for vortex extraction again. Centrifuge at 4000 rpm / min for 5 min. Take out the supernatant and dry it under nitrogen. Add 1 ml of 0.03 mol / L 2,3-dichloro-5,6-dicyano-1,4-benzoquinone acetonitrile solution to reconstitute the dried product. Shake for 10 min, add 0.2 g of neutral alumina for vortex extraction for 30 s, centrifuge at 10000 rpm / min for 10 min, and filter using a 0.22 μm PTFE needle filter membrane to obtain the sample extract.

[0061] 80 μL of the sample solution to be tested was vortexed with 100 μL of silver-coated gold nanorods for 1 min. 20 μL of 0.1 M NaCl was added and vortexed again. 10 μL of the mixture was transferred onto a glass slide using a pipette, forming spherical droplets. The slide was allowed to air dry for 10 min. Raman spectroscopy of the target area was performed using a portable Raman spectrometer under the following conditions: detection wavelength 785 nm, laser power 100%, integration time 10 s, and 3 integrations. A 1167 cm⁻¹ image was obtained. -1 Substitute the characteristic peak intensity at the given location into the above regression equation to calculate the malachite green concentration in Procambarus clarkii.

[0062] Example 2

[0063] A method for detecting malachite green veterinary drug residues in aquaculture water based on SERS using silver-coated gold nanorods is provided, comprising the following steps:

[0064] The preparation of silver-coated gold nanorods includes the following steps:

[0065] S21, Synthesis of gold seeds: Add 0.5 mL of 0.01 mol / L HAuCl4 solution to 2 mL of 0.2 mol / L hexadecyltrimethylammonium bromide (CTAB) solution, stir well, quickly add 0.4 mL of 0.01 mol / L NaBH4 solution in an ice bath, stir for 1 min, and then place the reaction solution in a 37℃ forced-air drying oven for constant temperature incubation for 1 h to obtain brown seed growth solution.

[0066] S22, Synthesis of Gold Nanorods: 1 mL of 0.01 mol / L AgNO3 was added to 10 mL of a binary surfactant solution composed of 0.05 M CTAB and 0.02 M sodium oleate (NaOL). The mixture was stirred at 500 rpm for 5 min, and then incubated at 37 °C for 15 min. Next, 10 mL of 0.001 mol / L HAuCl4 solution was added, and the mixture was stirred at 500 rpm for 90 min. The solution color changed from bright yellow to colorless. 1 mL of 1 mol / L HCl was added, and the mixture was stirred at 500 rpm for 15 min. Then, 50 μL of 0.1 mol / L ascorbic acid (L-AA) solution was added under vigorous stirring to obtain the growth solution. Finally, 80 μL of gold seed solution was added at 700 rpm and stirred for 1 min. The mixture was then incubated at 37 °C for 15 h. The prepared gold nanorods were centrifuged twice at high speed, the supernatant was removed, and ultrapure water was added to a final volume of 30 mL. After vortexing, the mixture was stored for later use.

[0067] S23, Synthesis of silver-coated gold nanorods: 80 μL of 0.01 mol / L AgNO3 was mixed with 500 μL of 0.1 M CTAC and incubated at 37 °C for 10 min. Then, 2 mL of the gold nanorods prepared in step S22 was added, and the mixture was incubated at 37 °C for 5 min. Next, 50 μL of 0.1 mol / L L-AA solution was added under vigorous stirring, and the resulting mixture was incubated at 37 °C for 4 h. The prepared silver-coated gold nanorods were centrifuged twice at 5000 rpm, brought to the original volume, and then purged with nitrogen as a protective agent. After vortex mixing, the mixture was stored for later use.

[0068] SERS detection of malachite green standard solution:

[0069] Take 10g of malachite green standard solution -10 -10 -6 100 μL of M and 100 μL of silver-coated gold nanorods were vortexed together, and 20 μL of 0.1 M NaBr was added and vortexed again. 10 μL of the mixture was transferred onto a glass slide using a pipette to form spherical droplets. The slide was allowed to dry naturally for 10 min. The area to be detected was then subjected to Raman spectroscopy using a portable Raman spectrometer. The test conditions were: detection wavelength of 785 nm, laser power of 100%, integration time of 10 s, and integration count of 3.

[0070] SERS spectral determination of malachite green and SERS analysis of standards:

[0071] A 793 cm⁻¹ was observed in the SERS spectrum of malachite green. -1 1167cm -1 1213cm -1 1293cm -1 1363cm -1 1391cm -1 1612cm -1 Based on the molecular structure of malachite green and the Raman peak assignment, the characteristic SERS peak at 1167 cm⁻¹ was determined. -1 The characteristic peak at a certain point is used as the discrimination criterion for SERS spectroscopy detection of malachite green. The intensity of the characteristic peak in the SERS spectrum of malachite green increases with the concentration of the standard solution by 10%. -10 -10 -6 The concentration of malachite green changes with M at 1167 cm⁻¹. -1 The linear regression equation for the characteristic peak intensity at a given location is: y = 19863.15logx + 176665.03, with a correlation coefficient R0. 2 =0.989.

[0072] Aquaculture water pretreatment:

[0073] Aquaculture water was filtered using a 0.22μm PTFE needle filter membrane to obtain sample extract.

[0074] 100 μL of the sample solution to be tested was vortexed with 100 μL of silver-coated gold nanorods for 1 min. 30 μL of 0.1 M NaBr was added and vortexed again. 10 μL of the mixture was transferred onto a glass slide using a pipette, forming a spherical droplet. The slide was allowed to dry for 10 min. Raman spectroscopy of the target area was performed using a portable Raman spectrometer under the following conditions: detection wavelength 785 nm, laser power 100%, integration time 10 s, and 3 integrations. A 1167 cm⁻¹ image was obtained. -1 Substituting the characteristic peak intensity at a given location into the linear regression equation above, the concentration of malachite green in aquaculture water is calculated.

[0075] Example 3

[0076] A method for detecting malachite green veterinary drug residues in fish samples based on SERS using silver-coated gold nanorods is provided, comprising the following steps:

[0077] The preparation of silver-coated gold nanorods includes the following steps:

[0078] S31, Synthesis of gold seeds: Add 0.5 mL of 0.01 mol / L HAuCl4 solution to 10 mL of 0.1 mol / L hexadecyltrimethylammonium bromide (CTAB) solution, stir well, quickly add 0.8 mL of 0.01 mol / L NaBH4 solution in an ice bath, stir for 3 min, and then place the reaction solution in a 37℃ forced-air drying oven for constant temperature incubation for 3 h to obtain brown seed growth solution.

[0079] S32, Synthesis of Gold Nanorods: 4 mL of 0.01 mol / L AgNO3 was added to 40 mL of a binary surfactant solution composed of 0.08 M CTAB and 0.02 M sodium oleate (NaOL). The mixture was stirred at 500 rpm for 5 min, and then incubated at 37 °C for 15 min. Next, 40 mL of 0.001 mol / L HAuCl4 solution was added, and the mixture was stirred at 700 rpm for 90 min. The solution color changed from bright yellow to colorless. 5 mL of 1 mol / L HCl was added, and the mixture was stirred at 400 rpm for 15 min. Then, 150 μL of 0.1 mol / L ascorbic acid (L-AA) solution was added under vigorous stirring to obtain the growth solution. Finally, 100 μL of gold seed solution was added at 700 rpm and stirred for 1 min, and the mixture was incubated at 37 °C for 15 h. The prepared gold nanorods were centrifuged twice at high speed to remove the supernatant. Ultrapure water was added to bring the volume to 30 mL, and the mixture was vortexed and stored for later use.

[0080] S33, Synthesis of Silver-Coated Gold Nanorods:

[0081] 200 μL of 0.01 mol / L AgNO3 was mixed with 600 μL of 0.08 M CTAC and incubated at 37 °C for 10 min. Then, 4 mL of the gold nanorods prepared in step S32 was added, and the mixture was incubated at 37 °C for 5 min. Next, 200 μL of 0.1 mol / L L-AA solution was added under vigorous stirring, and the resulting mixture was incubated at 37 °C for 4 h. The prepared silver-coated gold nanorods were centrifuged twice at 5000 rpm, brought to the original volume, and then purged with inert argon gas to remove oxygen from the solution. After vortex mixing, the mixture was stored for later use.

[0082] SERS detection of malachite green standard solution:

[0083] Take 10g of malachite green standard solution -10 -10 -6 100 μL of M was vortexed with 100 μL of silver-coated gold nanorods, and 50 μL of 0.1 M KI was added and vortexed again. 10 μL of the mixture was transferred onto a glass slide using a pipette to form spherical droplets. The slide was allowed to air dry for 10 min, and the area to be tested was then subjected to Raman spectroscopy using a portable Raman spectrometer. The test conditions were: detection wavelength of 785 nm, laser power of 100%, integration time of 10 s, and integration count of 3.

[0084] SERS spectral determination of malachite green and SERS analysis of standards:

[0085] A 793 cm⁻¹ was observed in the SERS spectrum of malachite green. -1 1167cm -1 1213cm -1 1293cm -1 1363cm -1 1391cm -1 1612cm -1 Based on the molecular structure of malachite green and the Raman peak assignment, the characteristic SERS peak at 1167 cm⁻¹ was determined. -1 The characteristic peak at a certain point is used as the criterion for SERS spectroscopy detection of malachite green. The intensity of the characteristic peak in the SERS spectrum of malachite green varies with the concentration of the standard solution (5 × 10⁻⁶). -9 M, 10 -8 M, 2×10 -8 M, 5×10 -8 M, 10 -8 M, 2×10 -7 The concentration of malachite green changes with M) and 1167 cm⁻¹. -1 The linear regression equation for the characteristic peak intensity at a given location is: y = 19863.15logx + 176665.03, with a correlation coefficient R0.2 =0.989.

[0086] Weigh 2.00g of homogenized fish meat sample and 0.5ml of 9.5g / L hydroxylamine hydrochloride into a 50ml capped plastic centrifuge tube. After standing for 10min, add 10ml of acetonitrile and 1.00g of anhydrous magnesium sulfate for vortex extraction. Add 4g of neutral alumina for vortex extraction again. Centrifuge at 4000rpm / min for 5min. Take out the supernatant and dry it under nitrogen. Add 1ml of 0.03mol / L 2,3-dichloro-5,6-dicyano-1,4-benzoquinone acetonitrile solution to reconstitute the dried product. Shake for 10min. Add 0.5g of neutral alumina for vortex extraction for 30s. Centrifuge at 12000rpm / min for 10min. Filter using a 0.22μm PTFE needle filter membrane to obtain the sample extract.

[0087] 100 μL of the sample solution to be tested was vortexed with 100 μL of silver-coated gold nanorods for 1 min. 50 μL of 0.1 M KI was added and vortexed again. 10 μL of the mixture was transferred onto a glass slide using a pipette, forming spherical droplets. The slide was allowed to air dry for 10 min. Raman spectroscopy of the target area was performed using a portable Raman spectrometer under the following conditions: detection wavelength 785 nm, laser power 100%, integration time 10 s, and 3 integrations. A 1167 cm⁻¹ image was obtained. -1 Substituting the characteristic peak intensity at a given location into the linear regression equation y = 19863.15logx + 176665.03, the correlation coefficient R0 is calculated. 2 =0.989, calculate the concentration of malachite green in the fish meat sample.

[0088] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for detecting malachite green veterinary drug residues based on SERS using silver-coated gold nanorods, characterized in that, include: Silver-coated gold nanorods were provided as Raman scattering enhancers and as a standard solution of malachite green, the analyte. The malachite green standard solution was mixed with the silver-coated gold nanorods, and a coagulating agent was added to form a first mixed solution. The first mixed solution was subjected to Raman spectroscopy to obtain the surface-enhanced Raman scattering (SERS) peaks of the malachite green standard solution. The characteristic peaks used to identify malachite green by SERS were determined by using the molecular structure of malachite green and the Raman peak positions. The linear relationship between the concentration of malachite green and the peak intensity of the characteristic peaks was also determined. The sample solution to be tested is mixed with the silver-coated gold nanorods, and then a coagulant is added to form a second mixed solution. The second mixed solution is subjected to Raman spectroscopy to obtain the peak intensity of the characteristic peak of malachite green in the sample solution to be tested. The concentration of malachite green in the sample solution to be tested is calculated by the linear relationship between the concentration of malachite green and the peak intensity of the characteristic peak, that is, the residual amount of malachite green is obtained. The coagulant is any one of NaCl, KI, Na2SO4, and NaBr; The method further includes preparing the silver-coated gold nanorods: S1, Synthesis of gold seeds: Mix 0.1 mL - 1.0 mL of 0.01 mol / L HAuCl4 solution with 1 mL - 12 mL of 0.1 mol / L hexadecyltrimethylammonium bromide solution to obtain a first solution. Then, quickly add 0.1 mL - 0.8 mL of 0.01 mol / L NaBH4 solution in an ice bath to the first solution to form a first reaction solution. After incubating the first reaction solution at a constant temperature, a brown gold seed solution is obtained. S2, Synthesis of Gold Nanorods: 1 mL - 4 mL of 0.01 mol / L AgNO3 was mixed thoroughly with a binary surfactant solution consisting of 10 mL - 80 mL of 0.08 M hexadecyltrimethylammonium bromide and 0.02 M sodium oleate to form a second solution. This second solution was incubated at a constant temperature. Then, 10 mL - 40 mL of 0.001 mol / L HAuCl4 solution was added to the incubated second solution. After stirring at 500 rpm - 1000 rpm for 20 min - 100 min, the solution color changed from bright yellow to colorless. 1 mL - 5 mL of 1 mol / L HCl was added, and after stirring for 5 min - 20 min, the solution was further incubated under vigorous stirring. Ascorbic acid solution was used to obtain a growth solution, which was stirred at 300-800 rpm. The gold seed solution is added to the growth solution to obtain a second reaction solution. After the second reaction solution is incubated at a constant temperature and then centrifuged, the gold nanorods are obtained. S3, Synthesis of silver-coated gold nanorods: 0.01 mol / L AgNO3 and 0.08 M hexadecyltrimethylammonium chloride was mixed evenly, and after constant temperature incubation, 2 mL-4 mL of the gold nanorods prepared in step S2 were added. After further constant temperature incubation, the mixture was added under vigorous stirring. The ascorbic acid solution was used to obtain a mixed solution, which was then incubated at a constant temperature and centrifuged to obtain the silver-coated gold nanorods. The prepared silver-coated gold nanorod solution was protected with inert gas or nitrogen to remove dissolved oxygen and then stored in a sealed container.

2. The method for detecting malachite green veterinary drug residues based on SERS using silver-coated gold nanorods according to claim 1, characterized in that, The mass percentages of the sample solution to be tested, the silver-coated gold nanorod reinforcing agent, and the coagulant are 10:5-10:1-3.

3. The method for detecting malachite green veterinary drug residues based on SERS using silver-coated gold nanorods according to claim 1, characterized in that, It also includes: pretreating the sample to prepare the test sample solution: The sample and hydroxylamine hydrochloride were placed in a centrifuge tube and allowed to stand for 5-12 minutes. Then, acetonitrile and anhydrous magnesium sulfate were added for vortex extraction. Neutral alumina was then added for vortex extraction, centrifuged for 4-10 minutes, and the supernatant was removed and dried under nitrogen to obtain the dried product. 2,3-dichloro-5,6-dicyano-1,4-benzoquinone acetonitrile solution was added to the dried product for redissolution, shaken for 2-12 minutes, and then neutral alumina was added for vortex extraction, centrifuged for 4-12 minutes, and filtered to obtain the sample solution to be tested.

4. The method for detecting malachite green veterinary drug residues based on SERS using silver-coated gold nanorods according to claim 1, characterized in that, The Raman spectroscopy detection was performed under the conditions of a wavelength of 785 nm, a laser power of 10%-100%, an integration time of 1 s-20 s, and 2-4 integration times. The surface-enhanced Raman scattering peak of the malachite green standard solution appeared at 1167 cm⁻¹. -1 1361 cm -1 1390 cm -1 1612 cm -1 Place.

5. The method for detecting malachite green veterinary drug residues based on SERS using silver-coated gold nanorods according to claim 4, characterized in that, The characteristic peak for detecting malachite green using surface-enhanced Raman scattering spectroscopy was determined to be at 1167 cm⁻¹. -1 Place.

6. The method for detecting malachite green veterinary drug residues based on SERS using silver-coated gold nanorods according to any one of claims 1-5, characterized in that, The method was used to detect malachite green residues in aquatic products, and the detection limit for malachite green residues in aquatic products was 1.58 × 10⁻⁶. -9 M.

7. The method for detecting malachite green veterinary drug residues based on SERS using silver-coated gold nanorods according to any one of claims 1-5, characterized in that, The concentration of malachite green was measured in the range of 5 × 10⁻⁶. -9 M - 2×10 -7 M.

8. The method for detecting malachite green veterinary drug residues based on SERS using silver-coated gold nanorods according to any one of claims 1-5, characterized in that, The amount of the silver-coated gold nanorods added is .

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