Method for detecting malachite green on basis of Antisine / GO / Ag SERS (Surface Enhanced Raman Scattering) substrate prepared by electro-deposition

Abstract

The invention discloses a malachite green detection method based on an Antigen / GO / Ag SERS substrate prepared by electrodeposition. The method comprises the following steps: preparing the substrate based on an electrodeposition method; the method comprises the following steps: dissolving malachite green in a phosphate buffer solution to prepare a standard solution, and diluting the prepared standard solution into sample solutions with different concentrations; setting detection parameters based on a surface enhanced Raman scattering technology, and carrying out Raman spectrum test on the sample solutions with different concentrations to obtain a test result; the composite structure of the substrate, the sensitivity and detection limit of the substrate to malachite green and the surface deposition condition of the substrate are used for verifying the effectiveness of the substrate; a real sample is prepared, a real sample solution is obtained, the real sample solution is analyzed based on the effective Anillin / GO / Ag SERS substrate, and a detection result of malachite green is obtained; the Aniline / GO / Ag thin film is adopted as an SERS detection substrate, the detection limit is low, and the stability is high.

Description

Technical Field

[0001] The present invention relates to the field of SERS spectral detection technology, and more specifically, to a malachite green detection method based on an Aniline / GO / Ag SERS substrate prepared by electrodeposition. Background Art

[0002] Malachite green (MG) residues in seafood pose a serious threat to human health. MG is a banned drug frequently detected in aquatic products. Due to its low price and anti-inflammatory and antibacterial properties, fishermen often use it as an antimicrobial agent to improve the survival rate of farmed organisms. However, consuming food containing MG residues can cause poisoning, shock, and increase the risk of cancer. Therefore, food safety testing of marine organisms is a critical issue.

[0003] Traditional toxicological testing methods include high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), electrochemical detection, fluorescence detection, and colorimetry. These methods require expensive equipment, complex procedures, and specialized technicians. Therefore, the development of a highly efficient, sensitive, and easy-to-use detection technology is urgently needed.

[0004] Surface-enhanced Raman scattering (SERS) technology leverages the localized surface plasmon resonance effect of metal nanostructures or specific materials to increase the Raman signal intensity of adsorbed molecules by over a million times. This significantly overcomes the low sensitivity bottleneck of traditional Raman spectroscopy and is widely used in trace substance detection, biomolecular identification, and environmental pollutant monitoring. SERS technology can fingerprint molecules during the non-destructive testing of chemical residues in food, and the Raman signal of the analyzed substance can be amplified by several orders of magnitude. SERS detection technology has outstanding potential in the field of analytical testing due to its high specificity, fast testing speed, high sensitivity, and low cost.

[0005] SERS sensors are currently fabricated using metal substrates, particularly Au, Ag, and Cu. While metal SERS substrates have high enhancement factors, they are expensive, chemically unstable, and exhibit poor SERS signal reproducibility. Furthermore, heavy metals are toxic, and the food being tested cannot be recycled. Subsequently, semiconductor SERS substrates, represented by ZnO, Si, TiO2, Ge, GP, and GO, began to replace metal SERS substrates. Although semiconductor substrates have weaker SERS performance than metal substrates, they offer advantages such as low cost, good biocompatibility, and strong chemical stability.

[0006] Therefore, there is an urgent need to develop new SERS substrate materials and structural design methods that combine high sensitivity, excellent stability, uniform signal response, strong corrosion resistance, and low cost. This is the key to solving the problem of malachite green (MG) residues in marine food.

[0007] At present, the preparation of SERS substrates mainly includes: spin coating, self-assembly, magnetron sputtering and electrodeposition. Among them, electrodeposition is a new preparation method with simple operation and the surface of the substrate obtained by electroplating is uniform. Electrodeposition is a technology that deposits metal or compound films on the surface of a conductive substrate through electrochemical control. Among them, graphene oxide (hereinafter referred to as GO) as a conductive substrate material for semiconductor SERS has good water solubility, especially rich oxygen-containing functional groups, and can be used to detect ions, small molecules, proteins and DNA. In the existing technology, GO is mostly used to prepare semiconductors by electrodeposition. In the process of detecting crystal violet, since the surface of GO has negatively charged (-COOH) functional groups and the surface has a large porosity and specific surface area, and crystal violet is a cationic molecule, GO can complete the adsorption of MG in a short time. This makes GO material have the advantage of being the most sensitive and most obvious in adsorbing MG.

[0008] In the prior art, the preparation of GO substrates mostly uses electrochemical stripping to improve conductivity. A method for preparing highly dispersible graphene oxide CN119430165A suppresses the shedding of graphite sheets by regulating the electrolyte and voltage parameters of the ratio of concentrated sulfuric acid and hydrogen peroxide. In addition, the uneven local current density during the electrochemical stripping process can easily lead to excessive oxidation of the edges of the GO sheets, forming structural defects and affecting the conductive stability of the substrate. In order to improve the interfacial bonding strength between GO and the substrate, the prior art often uses chemical stripping to strengthen the bonding effect. A method for stripping graphene oxide nanofilm from a substrate CN112744810B proposes the use of hydroiodic acid (HI) vapor to strip the GO film from a rigid porous substrate (such as anodized aluminum), but the high toxicity and easy residual properties of HI require a high-cost sealing system. In addition, the uneven coating problem caused by insufficient GO dispersibility is the main reason why the prior art relies on surfactants or ultrasonic dispersion processes. (High-concentration graphene slurry and preparation method and device thereof, CN107161993B) By adding expanded graphite and dispersant (polyvinyl pyrrolidone) in stages, the slurry uniformity is improved, the ultrasonic exfoliation efficiency is optimized, and agglomeration is avoided. However, the problem of secondary agglomeration during the drying process forces the process to be complicated. Summary of the Invention

[0009] In order to solve at least one of the above technical problems, the present invention proposes a malachite green detection method based on an Aniline / GO / Ag SERS substrate prepared by electrodeposition.

[0010] The first aspect of the present invention provides a method for detecting malachite green based on an Aniline / GO / Ag SERS substrate prepared by electrodeposition, comprising the following steps:

[0011] Aniline / GO / Ag SERS substrate, i.e. aniline / graphene oxide / silver substrate, was prepared based on electrodeposition;

[0012] Dissolve malachite green in phosphate buffer solution to prepare a standard solution, and dilute the prepared standard solution to prepare sample solutions of different concentrations;

[0013] The detection parameters were set based on surface-enhanced Raman scattering technology, and Raman spectroscopy tests were performed on sample solutions of different concentrations to obtain the test results;

[0014] Based on the test results, the composite structure of the Aniline / GO / Ag SERS substrate, the substrate's sensitivity and detection limit to malachite green, and the deposition of Aniline and GO on the substrate surface were analyzed to verify the effectiveness of the Aniline / GO / Ag SERS substrate.

[0015] Real samples were prepared to obtain real sample solutions, which were then analyzed based on the validated Aniline / GO / Ag SERS substrate to obtain the detection results of malachite green.

[0016] In a preferred embodiment of the present invention, the method for preparing an Aniline / GO / Ag SERS substrate includes:

[0017] A mixed solution C with a volume of 30 mL was formed by mixing solutions A and B, and then deposited on a stainless steel substrate by electroplating;

[0018] Solid particles D and solid particles E are mixed and deionized water is added to form 25 mL to 35 mL of a mixed solution F. The substrate after deposition is taken out and electrodeposition is continued in solution F.

[0019] The solution A is the conductive agent aniline; the solution B is graphene oxide; the mixed solution C contains 0.5ml-0.7ml of the conductive agent aniline and 28.5ml-29.3ml of graphene oxide; the solid particles D are silver nitrate AgNO3, the solid particles E are citric acid, and the solution F contains 0.04g-0.06g of silver nitrate AgNO3 and 0.5g-0.54g of citric acid.

[0020] In a preferred embodiment of the present invention, an Aniline / GO / Ag SERS substrate is prepared based on an electrodeposition method, specifically comprising:

[0021] Soak a piece of stainless steel sheet in deionized water and clean it with an ultrasonic cleaner for 20-30 minutes to initially clean the contaminants. Take out the sheet and soak it in anhydrous ethanol. Clean it with an ultrasonic cleaner and then take it out to air dry.

[0022] Prepare a mixed solution of 0.5-1 mol / L sulfuric acid and 0.2 mol / -0.25 mol / L aniline, sonicate for 5-10 minutes, and then add graphene oxide to form a mixed solution with a concentration of 0.03 g / L-0.08 g / L;

[0023] Using stainless steel as the working electrode and a platinum column as the counter electrode, Aniline / GO substrate was obtained by deposition at a constant voltage of 6 V in the prepared mixed solution for 20-30 minutes.

[0024] A solution containing 0.05 mol / L-0.1 mol / L citric acid and 0.01 mol / L-0.015 mol / L silver nitrate was prepared, stainless steel was used as a working electrode, a platinum column was used as a counter electrode, and electrodeposition was performed at a potential of -1 V for 20-30 minutes to prepare an Aniline / GO / Ag composite film;

[0025] After the electrodeposition, the Aniline / GO / Ag composite film was rinsed with deionized water for 4–5 min to remove non-polymerized electrolyte and other surface residues.

[0026] In a preferred embodiment of the present invention, the real sample is a tilapia fillet, and the steps for preparing the real sample are:

[0027] The fish fillets were minced, homogenized, and frozen for analysis. The thawed tissue samples were spiked with the appropriate concentration of analyte, stored at room temperature for 20–30 min, protected from light, and thoroughly mixed before extraction.

[0028] To the fortified sample, 1 mL-2 mL of ammonium acetate buffer at pH 4.5, 1 mL-2 mL of 20% hydroxylamine hydrochloride solution (HAH), 0.5 mL-1 mL of 1 mol L-1 p-toluenesulfonic acid, and 3 mL-5 mL of acetonitrile were added;

[0029] Add 0.5g-1g of aluminum oxide, shake vigorously for 1min-2min, centrifuge for 3min-5min, mix the supernatant with 3ml-5ml of dichloromethane, and centrifuge at 6500rpm-7000rpm for 3min-5min;

[0030] Transfer the dichloromethane layer to a flask and dry it at 40-45°C;

[0031] The dried residue was redissolved in 1 mL-2 mL of methanol and diluted to different concentrations with PBST.

[0032] In a preferred embodiment of the present invention, the detection parameters include an excitation wavelength of 532 nm, a detection light source of a He-Ne laser source, a laser source power of 10 to 30 mW, and a spectrum acquisition wave number of 500 to 2000 cm -1 .

[0033] In a preferred embodiment of the present invention, the test results include the sensitivity and detection limit of the substrate to MG, the authenticity of the substrate, and the stability of the substrate.

[0034] In a preferred embodiment of the present invention, the method for detecting the stability of the substrate includes: storing the substrate at 20° C.-24° C. for 20-30 days, performing SERS testing every 3-5 days, and analyzing changes in signal intensity.

[0035] In a preferred embodiment of the present invention, the concentration of the sample solution is 0.01 μM-1 μM.

[0036] In a preferred embodiment of the present invention, the dilution concentration range of the residue after being dissolved in methanol is 10 -12 nM-10 - 8 nM.

[0037] The above technical solution of the present invention has the following advantages over the prior art:

[0038] This application achieves strong SERS enhancement capability through the Aniline / GO / Ag substrate. Since aniline molecules can be easily adsorbed on the graphene oxide surface through electrostatic adhesion and π interaction, an Aniline / GO film is formed on the surface of a stainless steel sheet by electrodeposition. In addition, since the surface of graphene oxide has rich functional groups, it can effectively adsorb the molecules to be tested. Secondly, during the electrodeposition process, the SERS performance of the substrate is further improved by adding AgNO3 solution. The formation of AgNPs on the film surface can enhance the contribution of physical enhancement in SERS. The sensitivity, recovery rate and stability of the substrate were evaluated by using malachite green (MG) as a probe to improve the detection effectiveness.

[0039] The Aniline / GO / Ag film used as the SERS detection substrate offers advantages such as low detection limits and strong stability. Under 532nM (2.33eV) laser excitation, the substrate is capable of detecting MG residues at concentrations of 10-12M. Furthermore, real-world sample testing using fish fillets demonstrated recoveries of 96%-107% within the substrate's detection limit. Even after 30 days of storage, the substrate retained 80% of its initial signal strength, demonstrating high stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific embodiments or the description of the prior art. Obviously, some of the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0041] Figure 1 This is a diagram showing the preparation principle of the Aniline / GO / Ag substrate and the actual sample testing process according to an embodiment of the present invention;

[0042] Figure 2 This is an EDS elemental analysis spectrum of the Aniline / GO / Ag substrate according to an embodiment of the present invention;

[0043] Figure 3 is a Raman spectrum of the Aniline / GO / Ag substrate and MG molecules in an embodiment of the present invention;

[0044] Figure 4 2. EIS graphs of Aniline, Aniline / GO, and Aniline / GO / Ag substrates in PBS buffer according to an embodiment of the present invention;

[0045] Figure 5 This is a Raman spectrum and sensitivity curve of MG on an Aniline / GO / Ag substrate under 532nM laser in an embodiment of the present invention;

[0046] Figure 6 This is a Raman spectrum and recovery graph of MG residual in fish tissue detected by Aniline / GO / Ag substrate under 532nM laser in an embodiment of the present invention;

[0047] Figure 7 This is a trend chart of the performance degradation of the Aniline / GO / Ag substrate in an embodiment of the present invention within 30 days. DETAILED DESCRIPTION

[0048] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described in detail below in conjunction with specific embodiments. It should be noted that, in the absence of conflict, the embodiments of the present application and the features therein can be combined with each other.

[0049] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the present invention is not limited to the specific embodiments disclosed below.

[0050] Example 1

[0051] See also Figure 1-Figure 7 As shown, the present invention proposes a method for preparing an Aniline / GO / Ag substrate SERS substrate by electrodeposition to detect malachite green. Figure 1 As shown in the figure, the preparation principle of Aniline / GO / Ag substrate was studied. In this example, aniline was deposited on graphene oxide through the π stacking interaction and hydrogen bonding between aniline molecules and graphene oxide skeleton, as shown in the figure. Figure 1 As shown. A stainless steel sheet was used as the working electrode. Under a positive voltage, Aniline / GO was eventually attracted and adhered to the surface of the stainless steel sheet. During the electrodeposition process, AgNO3 and citric acid were added to the substrate for secondary deposition. Ag, as a common precious metal, can significantly improve the SERS performance of the substrate. The addition of citric acid can improve the uniformity of the deposition and further enhance the SERS enhancement ability of the substrate. Finally, MG was used as a probe to evaluate the sensitivity, recovery rate and stability of the substrate, and the effectiveness of the substrate was verified in real samples.

[0052] A novel method for preparing a highly efficient surface-enhanced Raman scattering substrate, aniline / graphene oxide / silver (Aniline / GO / Ag) substrate, is described. The method comprises the following steps:

[0053] Solutions A and B were mixed to form a 30 mL mixed solution C, which was then deposited on a stainless steel substrate by electroplating. Solid particles D and solid particles E were mixed and deionized water was added to form a 30 mL mixed solution F. The substrate after deposition was removed and electrodeposition continued in solution F.

[0054] Solution A is the conductive agent aniline (hereinafter referred to as Anil); solution B is graphene oxide (hereinafter referred to as GO); mixed solution C contains 0.7ml of Anil and 29.3ml of GO (concentration is 1g / L); solid particle D is silver nitrate (AgNO3); solid particle E is citric acid; solution F contains 0.06g of AgNO3 and 0.54g of citric acid.

[0055] Aniline / GO / Ag substrate step, preferably:

[0056] S1: Soak a stainless steel sheet in deionized water and clean it in an ultrasonic cleaner for 30 minutes to initially remove contaminants. Next, remove the sheet and soak it in anhydrous ethanol and clean it in an ultrasonic cleaner for 30 minutes. Then, remove it and let it air dry.

[0057] S2: Prepare a mixed solution of 1 mol / L sulfuric acid and 0.25 mol / L aniline, add graphene oxide after ultrasonic treatment for 10 minutes to form a mixed solution with a concentration of 0.05 g / L.

[0058] S3: Using stainless steel as the working electrode and a platinum column as the counter electrode, Aniline / GO substrate was obtained by deposition at a constant voltage of 6 V for 30 minutes in the mixed solution configured in S2.

[0059] S4: Next, a solution containing 0.1 mol / L citric acid and 0.015 mol / L silver nitrate was prepared. Using stainless steel as the working electrode and a platinum column as the counter electrode, electrodeposition was performed at a potential of -1 V for 30 minutes to produce an Aniline / GO / Ag composite film.

[0060] S5: After the electrodeposition, the Aniline / GO / Ag composite film was rinsed with deionized water for 4 min to remove the non-polymerized electrolyte and other surface residues.

[0061] The elemental analysis of the obtained Aniline / GO / Ag substrate is shown in the figure below: Figure 2 As shown. Figure 2 It can be seen that the Aniline / GO / Ag substrate in this example is mainly composed of Ag element.

[0062] The technical solution of the present invention also provides a method for detecting the MG content in fish tissue using an Aniline / GO / Ag SERS substrate, the steps of which include:

[0063] The MG sample solution preparation steps are preferably:

[0064] S1: Dissolve 0.195 g of MG in 30 mL of PBS to prepare a 0.01 M standard solution.

[0065] S2: Dilute the standard solution prepared in S1 into sample solutions of different concentrations (0.01μM, 0.05μM, 0.1μM, 0.3μM, 0.5μM, 0.7μM, 1μM).

[0066] The preparation steps of the real sample (tilapia fillet) are preferably:

[0067] S1: Fish fillets were minced, homogenized, and frozen for analysis. The thawed tissue samples were spiked with the appropriate concentration of analyte and stored at room temperature for 30 minutes, protected from light, and thoroughly mixed before extraction.

[0068] S2: 2 mL of ammonium acetate buffer (pH 4.5), 1 mL of 20% (w / w) hydroxylamine hydrochloride solution (HAH), 0.5 mL of 1 mol L-1 p-toluenesulfonic acid, and 5 mL of acetonitrile were added to the fortified sample.

[0069] S3: Add 1 g of alumina, shake vigorously for 1 min, and centrifuge at 1300 × g for 5 min. Mix the supernatant with 5 ml of dichloromethane and centrifuge at 7000 rpm for 5 min.

[0070] S4: The dichloromethane layer was transferred to a flask and dried at 45°C.

[0071] S5: The dried residue was redissolved in 1 mL of methanol and diluted with PBST to different concentrations (10 -12 ,10 -11 ,10 -10 ,10 -9 ,10 -8 nM).

[0072] The SERE detection method steps based on Aniline / GO / Ag substrate are preferably:

[0073] S1: The SERS detection excitation wavelength is selected from 532 nm. The SERS detection light source is a He-Ne laser source with a laser source power of 10-30 mW, preferably 10-15 mW. The wave number of the collected spectrum is 500-2000 cm -1 , and inspect the inspection board under the inspection conditions of the connection material microscope.

[0074] S2: In order to further analyze whether the Aniline / GO / Ag SERS substrate forms a composite structure, the EDS Raman spectrum of the substrate was measured. It can be clearly found that there is Ag element on the surface of the film. The results show that AgNPs have been successfully deposited to form the surface of the Aniline / GO substrate.

[0075] S3: To further investigate the deposition of Aniline and GO on the substrate surface, we used Raman spectroscopy to test the Aniline / GO / Ag substrate and MG molecules, respectively. Figure 3 As shown. For MG, 811cm -1 ,923cm -1 and 1186cm -1 The Raman peak at 1372 cm belongs to the CH bend. -1 and 1401cm -1 Symmetric stretching vibration of CN. 1600cm -1 ,1626cm -1 The Raman peaks at 1345 cm-1 belong to the stretching vibration of the ring CC group. -1 and 1588cm -1 The Raman peaks at 1165cm belong to the D peak and G peak of GO, which represent the disorder vibration induced by defects and the lattice vibration of the graphite structure itself.-1 Belongs to the CH plane bending vibration in Anil. 1253cm -1 Belongs to the stretching of CN in the polaron unit in Anil. 1377cm -1 Belongs to Anil's CC stretch.

[0076] S4: Figure 4 The EIS results in Figure 2 show that the Rct of the Aniline / GO film is the highest, while the Rct of the Aniline / GO / Ag substrate is the lowest. This further demonstrates that the Aniline / GO / Ag substrate has strong electrochemical activity, facilitating charge transfer between the substrate and the MG molecules, thereby enhancing the contribution of chemical enhancement to SERS. Furthermore, the deposition of GO nanosheets is motivated by their large surface area, providing more deposition sites for AgNP detection, thereby improving the SERS performance of the substrate.

[0077] S5: If Figure 5 As shown in the figure, the sensitivity and detection limit of the detection substrate to MG were tested by Raman spectroscopy on MG solutions with different concentrations. Figure 5 (a) means in 10 -8 M-10 -12 EC-SERS detection results of MG molecules by the substrate in the range of 10-12 M. When the solution concentration is as low as 10-12 M, the basic peak morphology of MG can also be seen, which shows that the substrate not only has the function of fingerprint recognition for MG molecules, but is also suitable for ultra-low concentration detection. -1 The peak at is studied as the characteristic peak of MG. Figure 5 As shown in (b), with the increase of MG concentration, the 1600 cm -1 The slope of the fitted line between MG concentration and characteristic peak intensity is -3938.8, indicating that the substrate has a strong sensitivity to MG molecules.

[0078] S6: To test the authenticity of the substrate, Raman spectroscopy was performed on fish tissue solutions containing different concentrations of MG. The substrate was immersed in the prepared real sample solution and taken out after 60 minutes. Figure 6 (a) shows 1600 cm -1 The characteristic peak at is more obvious, and the SERS substrate can be used at a low temperature of 1×10 -11 MG molecules were successfully detected at a concentration of M. In addition, Figure 6 As shown in (b), the recovery rate of MG was analyzed. -8 M-10 -12 The recovery rate range between M was 96%-107%, indicating that the substrate had high reliability and accuracy.

[0079] S7: To test the stability of the substrate, the substrate was stored at 24°C for two weeks to evaluate its stability, and SERS tests were performed every five days. -1 The peak value at the 4th day is used as the denominator A, and the peak values obtained from the tests on the remaining days are used as the numerator B, and the retained signal intensity ratio B / A is calculated. Figure 7 In the experiment, after 30 days of storage, the substrate still retained 80% of the initial signal intensity, which indicated that the sensor had good stability.

[0080] The technical features of the above embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0081] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein, but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

[0082] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for detecting malachite green based on an Aniline / GO / Ag SERS substrate prepared by electrodeposition, characterized in that: The steps include: Aniline / GO / Ag SERS substrate, i.e. aniline / graphene oxide / silver substrate, was prepared based on electrodeposition; Dissolve malachite green in phosphate buffer solution to prepare a standard solution, and dilute the prepared standard solution to prepare sample solutions of different concentrations; The detection parameters were set based on surface-enhanced Raman scattering technology, and Raman spectroscopy tests were performed on sample solutions of different concentrations to obtain the test results; Based on the test results, the composite structure of the Aniline / GO / Ag SERS substrate, the substrate's sensitivity and detection limit to malachite green, and the deposition of Aniline and GO on the substrate surface were analyzed to verify the effectiveness of the Aniline / GO / Ag SERS substrate. Real samples were prepared to obtain real sample solutions, which were then analyzed based on the validated Aniline / GO / Ag SERS substrate to obtain the detection results of malachite green.

2. The method for detecting malachite green based on an Aniline / GO / Ag SERS substrate prepared by electrodeposition according to claim 1, characterized in that: The preparation method of Aniline / GO / Ag SERS substrate includes: A mixed solution C with a volume of 30 mL was formed by mixing solutions A and B, and then deposited on a stainless steel substrate by electroplating; Mix solid particles D and solid particles E and add deionized water to form 25 mL-35 mL of mixed solution F. Remove the deposited substrate and continue electrodeposition in solution F. The solution A is the conductive agent aniline; the solution B is graphene oxide; the mixed solution C contains 0.5ml-0.7ml of the conductive agent aniline and 28.5ml-29.3ml of graphene oxide; the solid particles D are silver nitrate AgNO3, the solid particles E are citric acid, and the solution F contains 0.04g-0.06g of silver nitrate AgNO3 and 0.5g-0.54g of citric acid.

3. The method for detecting malachite green based on an Aniline / GO / Ag SERS substrate prepared by electrodeposition according to claim 1, characterized in that: Aniline / GO / Ag SERS substrate was prepared based on the electrodeposition method, specifically including: Soak a piece of stainless steel sheet in deionized water and clean it with an ultrasonic cleaner for 20-30 minutes to initially clean the contaminants. Take out the sheet and soak it in anhydrous ethanol. Clean it with an ultrasonic cleaner and then take it out to air dry. Prepare a mixed solution of 0.5-1 mol / L sulfuric acid and 0.2 mol / -0.25 mol / L aniline, sonicate for 5-10 minutes, and then add graphene oxide to form a mixed solution with a concentration of 0.03 g / L-0.08 g / L; Using stainless steel as the working electrode and a platinum column as the counter electrode, Aniline / GO substrate was obtained by deposition at a constant voltage of 6 V in the prepared mixed solution for 20-30 minutes. A solution containing 0.05 mol / L-0.1 mol / L citric acid and 0.01 mol / L-0.015 mol / L silver nitrate was prepared, stainless steel was used as a working electrode, a platinum column was used as a counter electrode, and electrodeposition was performed at a potential of -1 V for 20-30 minutes to prepare an Aniline / GO / Ag composite film; After the electrodeposition, the Aniline / GO / Ag composite film was rinsed with deionized water for 4–5 min to remove the non-polymerized electrolyte and other surface residues.

4. The method for detecting malachite green based on an Aniline / GO / Ag SERS substrate prepared by electrodeposition according to claim 1, characterized in that: The real sample is a tilapia fillet, and the preparation steps of the real sample are as follows: The fish fillets were minced, homogenized, and frozen for analysis. The thawed tissue samples were spiked with the appropriate concentration of analyte, stored at room temperature for 20–30 min, protected from light, and thoroughly mixed before extraction. To the fortified sample, 1 mL-2 mL of ammonium acetate buffer at pH 4.5, 1 mL-2 mL of 20% hydroxylamine hydrochloride solution (HAH), 0.5 mL-1 mL of 1 mol L-1 p-toluenesulfonic acid, and 3 mL-5 mL of acetonitrile were added; Add 0.5g-1g of aluminum oxide, shake vigorously for 1min-2min, centrifuge for 3min-5min, mix the supernatant with 3ml-5ml of dichloromethane, and centrifuge at 6500rpm-7000rpm for 3min-5min; Transfer the dichloromethane layer to a flask and dry it at 40-45°C; The dried residue was redissolved in 1 mL-2 mL of methanol and diluted to different concentrations with PBST.

5. The method for detecting malachite green based on an Aniline / GO / Ag SERS substrate prepared by electrodeposition according to claim 1, characterized in that: The detection parameters include an excitation wavelength of 532 nm, a He-Ne laser source with a power of 10 to 30 mW, and a spectrum acquisition wave number of 500 to 2000 cm -1 .

6. The method for detecting malachite green based on an Aniline / GO / Ag SERS substrate prepared by electrodeposition according to claim 1, characterized in that: The test results include the sensitivity and detection limit of the substrate to MG, the authenticity of the substrate and the stability of the substrate.

7. The method for detecting malachite green based on an Aniline / GO / Ag SERS substrate prepared by electrodeposition according to claim 1, characterized in that: The stability detection method of the substrate includes: storing the substrate at 20° C.-24° C. for 20-30 days, performing SERS testing every 3-5 days, and analyzing changes in signal intensity.

8. The method for detecting malachite green based on an Aniline / GO / Ag SERS substrate prepared by electrodeposition according to claim 1, characterized in that: The concentration of the sample solution is 0.01 μM-1 μM.

9. The method for detecting malachite green based on an Aniline / GO / Ag SERS substrate prepared by electrodeposition according to claim 4, characterized in that: The dilution range of the residue after dissolving in methanol was 10 -12 nM-10 -8 nM.

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