Preparation method of a self-assembled flexible SERS substrate and its application in pesticide residue detection

Abstract

The present invention relates to the technical field of fruit and vegetable pesticide residue detection, specifically a method for preparing a self-assembled flexible SERS substrate and its application in pesticide residue detection, wherein the flexible SERS substrate is composed of AB silica gel as a flexible substrate and Au@Ag nanorods self-assembled into a two-dimensional nanofilm composite; firstly, the Au nanorods are prepared by a seed method, and then silver nanoparticles are wrapped in the outer layer, and Au@Ag is obtained by a liquid-liquid interface self-assembly method, and then A and B glues are mixed in a ratio of 1:1 and solidified into a transparent silica gel-based film, and the Au@Ag film is printed and dyed and coated on the transparent silica gel-based film, that is, assembled into a flexible SERS substrate. The flexible SERS substrate of the present application has good plasticity, excellent flexibility and negligible SERS background signal, can be attached to the surface of an actual sample for detection, has a simple, convenient and operable practical application prospect, and provides a new method and technology for on-site rapid detection of fruit and vegetable pesticide residues.

Description

Technical Field

[0001] The invention relates to the technical field of fruit and vegetable pesticide residue detection, and in particular to a method for preparing a self-assembled flexible SERS substrate and an application thereof in pesticide residue detection. Background Art

[0002] Nowadays, pesticides are increasingly used in agriculture as chemical reagents to protect crops and fruits from diseases, pests and weeds. However, studies have shown that the vast majority of applied pesticides will eventually enter the environment, agricultural products and even the food chain. Even at trace levels, they may directly pollute the agricultural environment and cause health risks. Therefore, whether the food we eat is safe has become a current concern. Traditional methods for detecting pesticide residues include gas chromatography, liquid chromatography, enzyme-linked immunosorbent assay, etc. However, these traditional methods have more or less disadvantages such as complex operation, long detection time, expensive instruments and easy false positive results, which seriously hinder the rapid circulation of agricultural products in the market and affect people's table safety. Therefore, there is an urgent need for a rapid, sensitive, simple and economical analytical method to detect pesticide residues in fruits and vegetables.

[0003] As a powerful analytical tool, Raman spectroscopy can be used to analyze the composition of compounds. However, its own principle determines that it has poor sensitivity. With the development of nanotechnology, surface enhanced Raman (SERS) has gradually come into people's view. SERS technology can quickly and accurately provide fingerprint information of molecular structure and is widely used in environmental monitoring, biological science and food safety management. However, the instability of nanomaterials has seriously limited the application of SERS technology in practice. Therefore, many scientists are committed to assembling SERS substrates with more hotspots and more stable to improve the sensitivity of SERS detection. In order to obtain a more sensitive and stable SERS substrate, interfacial self-assembly technology is considered to be a simple, fast and economical method for preparing two-dimensional nanoarrays.

[0004] At present, the Chinese invention patent with publication number CN112986214A discloses a method for preparing a flexible substrate for quantitative detection of pesticide residues. Using polydimethylsiloxane, chloroauric acid and polyimide film as raw materials, a large-area graphene-gold particle composite flexible substrate is quickly prepared by two laser direct writing techniques; by using the chemical enhancement of graphene and the electromagnetic field enhancement of gold particles, the intensity of the graphene Raman peak in the SERS spectrum remains unchanged, and the intensity ratio of the pesticide residue Raman peak to the graphene Raman peak is used to achieve quantitative detection of pesticide residues on the surface of fruits and vegetables. However, there are problems such as complex preparation process and inconvenient detection, which limits the practical application of SERS in the detection of pesticide residues in fruits and vegetables.

[0005] The present invention adopts a liquid-liquid interface self-assembly method to self-assemble Au@Ag into a two-dimensional thin film, which is then directly assembled with a food-grade silica gel flexible substrate through "printing and dyeing" to obtain a flexible SERS substrate. The substrate has high and stable SERS activity, good flexibility and plasticity, and can be suitable for on-site rapid detection of pesticide residues in actual fruits and vegetables. Summary of the invention

[0006] In view of the low sensitivity and impractical application of current SERS detection, the present invention provides a method for preparing a flexible SERS substrate which is simple to prepare, convenient to operate and low in price, and introduces the application of the flexible SERS substrate in actual detection of pesticide residues in fruits and vegetables.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] The present invention discloses a method for preparing a self-assembled flexible SERS substrate, comprising the following steps:

[0009] S1. Preparation of Au@Ag nanorods

[0010] a. Prepare Au seed solution: Mix CTAB (hexadecyltrimethylammonium bromide) solution and tetrachloroauric acid solution under heating conditions, and stir evenly to form a golden solution; then add sodium borohydride solution prepared with ice water to form a dark brown solution, increase the stirring speed and continue stirring for 28-32 seconds, then age at 28-32°C for 2-3 hours, and store at 3-5°C for use.

[0011] b. Prepare Au growth solution: Dissolve CTAB and sodium oleate in ultrapure water at 45-55°C with stirring, wait until the temperature drops to 25-30°C, add silver nitrate solution, stir evenly and let stand at 25-30°C for 15-20 minutes; then add tetrachloroauric acid solution and continue stirring until the solution turns milky white, then adjust the pH to 4-5 and continue stirring for 10-20 minutes; then add ascorbic acid solution, increase the stirring speed and continue stirring for 28-32 seconds, then add Au seed solution to the above mixed solution, reduce the stirring speed and continue stirring for 50-70 seconds, and then grow at 25-30°C for 12-16 hours; after the reaction is completed, centrifuge and wash, and concentrate to obtain Au growth solution.

[0012] c. Preparation of Au@Ag nanorods:

[0013] CTAC (hexadecyltrimethylammonium chloride) is mixed with silver nitrate, incubated at 25-30°C for 8-12 minutes, and then the Au growth solution is added to the mixed solution. After mixing, the mixture is incubated at 25-30°C for 4-6 minutes, and then shaken for reaction at 28-32°C for 3-5 hours to obtain a brown-yellow product; excess CTAC is removed by centrifugation and washing, and the unwrapped Au nanorods are removed by centrifugation and washing again. After repeating 3-5 times, the product is resuspended in ultrapure water to obtain Au@Ag nanorods.

[0014] S2. Preparation of flexible substrate

[0015] The food-grade silicone precursor A and the curing agent B are uniformly mixed in a weight ratio of 1:1, and then cured at 70-90° C. for 5-10 minutes to obtain a flexible substrate.

[0016] S3. Assembly of flexible SERS substrate

[0017] a. Au@Ag nanorods self-assembly.

[0018] Au@Ag nanorods are placed in a container and n-hexane is added to form an oil-water interface. Ethanol is then injected into the system, and a self-assembled Au@Ag nanorod film is formed at the oil-water interface. When the upper organic solvent evaporates at room temperature, the self-assembly of the Au@Ag nanorods is completed.

[0019] b. Assembly of flexible substrate: The Au@Ag nanorod film is printed from a container using a flexible substrate and then dried at room temperature to obtain a self-assembled flexible SERS substrate.

[0020] Beneficial effects: First, Au seed particles are prepared using CTAB as a surfactant and NaBH4 as a reducing agent; then, Au nanorods are prepared using CTAB and sodium oleate as dual surfactants, with silver nitrate as a rod-shaped control variable and the reducing effect of ascorbic acid; then, Ag is wrapped on the surface of the Au nanorods using CTAC as a surfactant to obtain Au@Ag nanorods; a single-layer Au@Ag nanorod film is obtained by liquid-liquid interface self-assembly; then, a flexible transparent film is obtained by mixing and curing silica gel A and B, and the flexible transparent film has adhesion, and the single-layer Au@Ag nanorod film is covered thereon without the need for other modifications, thereby assembling into a flexible SERS substrate.

[0021] The present application utilizes a self-assembly method to print and dye a single-layer two-dimensional film of Au@Ag nanorods onto a silica gel substrate. The preparation process is simple, the raw materials are inexpensive, and the obtained substrate has good uniformity and high stability. The flexible SERS substrate has a negligible SERS background signal, and the Au@Ag flexible substrate has excellent enhancement activity, which makes the detection sensitivity high.

[0022] Preferably, in step S1-a, the concentration of the CTAB solution is 0.1-0.2 mol / L, the concentration of the tetrachloroauric acid solution is 0.05-0.1 mol / L, and the molar ratio of CTAB to tetrachloroauric acid is 1:0.25-0.5.

[0023] Preferably, in step S1-a, the CTAB solution and the tetrachloroauric acid solution are mixed in a water bath, and the water bath heating temperature is 28-32°C.

[0024] Preferably, in step S1-a, the stirring speed of the CTAB solution and the tetrachloroauric acid solution is 500-700 rpm, and the stirring speed after adding sodium borohydride is 1200-1500 rpm.

[0025] Preferably, in step S1-b, the mass ratio of CTAB to sodium oleate is 1:0.1-0.35.

[0026] Preferably, in step S1-b, the molar ratio of silver nitrate, tetrachloroauric acid and ascorbic acid is 1:1.6-3.3:0.0053-0.008.

[0027] Preferably, in step S1-b, the volume ratio of the Au seed solution to the mixed solution is 1:400-550.

[0028] Preferably, in step S1-b, the stirring speed after adding silver nitrate, tetrachloroauric acid, adjusting pH, and adding Au seed solution is 600-700 rpm; the stirring speed after adding ascorbic acid is 1200-1500 rpm.

[0029] Preferably, in step S1-c, the molar ratio of silver nitrate to CTAC is 1:24-120.

[0030] Preferably, in step S1-c, the volume ratio of the Au growth solution to the mixed solution is 1:0.32-0.4.

[0031] Preferably, the first centrifugation in step S1-c has a rotation speed of 6000-8000 rpm and a time of 12-17 min.

[0032] Preferably, the second centrifugation in step S1-c has a rotation speed of 4500-4700 rpm and a time of 12-17 min.

[0033] Preferably, the silica gel A in step S2 is food grade silica gel.

[0034] Preferably, in step S3-a, the volume ratio of n-hexane, Au@Ag nanorods, and ethanol is 1:3-5:1.5-2.

[0035] Preferably, in step S3-a, the Au@Ag nanorod self-assembled film is in the form of an Au@Ag nanorod array.

[0036] Another aspect of the present invention discloses the application of the self-assembled flexible SERS substrate in the detection of pesticide residues.

[0037] Preferably, the self-assembled flexible SERS substrate is placed on the surface of fruits and vegetables, and then the surface is left for 10 seconds, and then the surface is uncovered and detected using a Raman spectrometer.

[0038] Preferably, the Raman spectrometer is a portable Raman spectrometer, the detector excitation wavelength is 785 nm, and the laser power is 50 mW.

[0039] Preferably, the fruits and vegetables include apples, strawberries, and mushrooms, and the pesticide residues include thiram.

[0040] Beneficial effects: The flexible SERS substrate of the present invention has good plasticity and excellent flexibility, and can be folded and wiped flexibly on fruits and vegetables. The food-grade silica gel used is non-toxic and harmless, has few Raman characteristic peaks, and has little interference. When used in combination with a portable Raman instrument, it is particularly suitable as a flexible substrate for the detection of pesticide residues on the surface of fruits and vegetables. It has a practical application prospect of being simple, convenient and operable, and provides a new method and technology for the on-site rapid detection of pesticide residues in fruits and vegetables.

[0041] The advantages of the present invention are:

[0042] 1. The present application first uses CTAB as a surfactant and NaBH4 as a reducing agent to prepare Au seed particles; then uses CTAB and sodium oleate as dual surfactants, and prepares Au nanorods under the reducing effect of silver nitrate as a rod-shaped control variable and ascorbic acid; then uses CTAC as a surfactant to wrap Ag on the surface of the Au nanorods to obtain Au@Ag nanorods; obtains an Au@Ag nanorod monolayer film by a liquid-liquid interface self-assembly method; then mixes and solidifies food-grade silica gel precursors A and B to obtain a transparent film, which has adhesiveness, and directly prints and dyes the Au@Ag nanorod monolayer film on it without using other modifications or transfers, so as to assemble it into a flexible SERS substrate.

[0043] 2. This application utilizes a self-assembly method to directly coat the formed Au@Ag nanorod single-layer two-dimensional film on a silica gel substrate. The preparation process is simple, the raw materials are inexpensive, and the resulting substrate has good uniformity and high stability. The flexible SERS substrate has a negligible SERS background signal, and the Au@Ag flexible substrate has excellent enhancement activity, which makes the detection sensitivity high.

[0044] 3. The flexible SERS substrate of the present application has good plasticity and excellent flexibility, and can be folded and wiped flexibly on fruits and vegetables. The food-grade silica gel used is non-toxic and harmless, has few Raman characteristic peaks, and has little interference. It can be used in combination with a portable Raman instrument, and is particularly suitable as a flexible substrate for the detection of pesticide residues on the surface of fruits and vegetables. It has a practical application prospect that is simple, convenient and operational, and provides a new method and technology for the on-site rapid detection of pesticide residues in fruits and vegetables. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a transmission electron microscopy image of the Au@Ag nanorods prepared in Example 1 of the present application.

[0046] Figure 2 This is the UV-visible absorption spectrum of the Au@Ag nanorods prepared in Example 1 of the present application.

[0047] Figure 3 Schematic diagram of the self-assembly process of Au@Ag nanorods in Example 1 of the present application.

[0048] Figure 4 Schematic diagram of the assembly process of the flexible SERS substrate in Example 1 of the present application.

[0049] Figure 5 This is a SERS spectrum diagram of the SERS activity detection of the flexible substrate in Example 4 of the present application.

[0050] Figure 6 This is the SERS spectrum diagram of strawberry pesticide residue detection in Example 5 of the present application. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0053] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0054] Example 1

[0055] The present application discloses a method for preparing a self-assembled flexible SERS substrate, comprising the following steps:

[0056] S1. Preparation of Au@Ag nanorods

[0057] a. Prepare Au seed solution: Use ultrapure water to prepare 0.2 mol / L CTAB solution, 0.05 mol / L tetrachloroauric acid solution, and use ice water to prepare 10 mmol / L sodium borohydride solution.

[0058] In a 30°C water bath, 2.5 mL of 0.2 mol / L CTAB (hexadecyltrimethylammonium bromide) solution was mixed with 2.5 mL of 0.05 mol / L tetrachloroauric acid solution, and gently stirred at 700 rpm for 2 min. A golden solution was formed after uniform stirring. Then, sodium borohydride solution was quickly added, and the solution changed from yellow to brown. At this time, it was vigorously stirred at 1300 rpm for 30 s, and then aged in a 30°C oven for 2-3 h, and stored in a 4°C refrigerator for use.

[0059] b. Prepare Au growth solution: Dissolve 0.7g CTAB and 0.1234g sodium oleate in 25mL ultrapure water at 50°C with stirring. After the temperature drops to 30°C, add 1.5mL 10mmol / L silver nitrate solution, stir at 700rpm for 5min, and let stand in a 30°C oven for 15min; then add 25mL 1mmol / L tetrachloroauric acid solution, continue stirring at 700rpm for 30-90min until the solution turns milky white, then adjust the pH of the solution to 4.5 with 3mL 1m HCl, and continue stirring at 700rpm for 15min; then add 80μL 0.1 mol / L ascorbic acid solution was stirred vigorously at 1200 rpm for 30 s, then 100 μL of Au seed solution was added to the mixed solution, and the mixture was slowly stirred at 700 rpm for 60 s, and then grown in an oven at 30°C for 14 h; after the reaction, the mixture was centrifuged and washed, and concentrated to obtain the Au growth solution.

[0060] c. Preparation of Au@Ag nanorods:

[0061] 3 mL of 0.08 mol / L CTAC (hexadecyltrimethylammonium chloride) was mixed with 1 mL of 10 mmol / L silver nitrate, and incubated at 30°C for 10 min. After taking out, 12.5 mL of Au growth solution was added to the mixed solution, and the mixture was incubated at 30°C for 5 min. Then, the mixture was shaken at 30°C for 4 h to obtain a brown-yellow product. The mixture was centrifuged at 7000 rpm for 15 min to wash away excess CTAC. The mixture was centrifuged at 4600 rpm for 15 min to wash away unwrapped Au nanorods. After repeating this process three times, the product was subjected to electron microscopy scanning and UV-visible absorption spectroscopy analysis. The analysis results are shown in FIG. Figure 1, Figure 2 shown. Figure 1 The scanning electron microscope image of the product is Figure 1 The core-shell structure of Au@Ag nanorods can be seen in the figure. Figure 2 is the UV-visible spectrum of the product, in which two absorption peaks appear. The one near 510nm is the plasma resonance band of Au NRs along the short axis direction, called the transverse absorption peak; the one near 780nm is called the longitudinal absorption peak, which proves the successful synthesis of AuNRs; after the AuNRs are wrapped with a Ag shell, the one near 650nm is the longitudinal dipole plasma resonance, the one at 500nm is attributed to the transverse dipole plasma resonance, and the one at 400nm belongs to the octopole resonance mode, indicating that the prepared product is Au@Ag nanorods; finally, the product is resuspended in 1mL of ultrapure water for use.

[0062] S2. Preparation of flexible substrate

[0063] Food-grade silicone rubber A and curing agent B were uniformly mixed in a weight ratio of 1:1, and then cured at 80° C. for 8 minutes to obtain a flexible substrate.

[0064] S3. Assembly of flexible SERS substrate

[0065] a. Au@Ag nanorods self-assembly:

[0066] like Figure 3 As shown, 3 mL of Au@Ag nanorods were placed in a beaker, and then 1 mL of n-hexane was added to it to quickly form an oil-water interface; then 2 mL of ethanol was quickly injected into the system with a syringe. At this time, an Au@Ag nanorod array was quickly formed at the oil-water interface. When the upper organic solvent evaporated at room temperature, the self-assembly of the Au@Ag nanorods was completed.

[0067] b. Assembly of flexible substrate: Figure 4 As shown, use tweezers to clamp a corner of the flexible substrate, gently cover it on the Au@Ag nanorod array, and quickly pick it up. The Au@Ag nanorod array will be directly and firmly printed on the surface of the flexible substrate. After drying under natural conditions, a self-assembled flexible SERS substrate can be obtained.

[0068] Example 2

[0069] The present application discloses a method for preparing a self-assembled flexible SERS substrate, comprising the following steps:

[0070] S1. Preparation of Au@Ag nanorods

[0071] a. Prepare Au seed solution: Use ultrapure water to prepare 0.1 mol / L CTAB solution, 0.05 mol / L tetrachloroauric acid solution, and use ice water to prepare 10 mmol / L sodium borohydride solution.

[0072] In a 28°C water bath, 2.5 mL of 0.1 mol / L CTAB (hexadecyltrimethylammonium bromide) solution was mixed with 2.5 mL of 0.05 mol / L tetrachloroauric acid solution, and gently stirred at 600 rpm for 2 min. A golden solution was formed after uniform stirring. Then, sodium borohydride solution was quickly added, and the solution changed from yellow to brown. At this time, it was vigorously stirred at 1200 rpm for 32 s, and then aged in a 28°C oven for 2-3 h, and stored in a 3°C refrigerator for use.

[0073] b. Preparation of Au growth solution: 0.7g CTAB and 0.2468g sodium oleate were stirred and dissolved in 25mL ultrapure water at 45°C. After the temperature dropped to 28°C, 1.5mL 10mmol / L silver nitrate solution was added, stirred at 600rpm for 5min, and then allowed to stand in a 25°C oven for 20min. Then 50mL 1mmol / L tetrachloroauric acid solution was added, and stirring continued at 600rpm for 30-90min until the solution turned milky white. At this time, 3mL 1m HCl was added to adjust the pH of the solution to 4, and stirring continued at 600rpm for 20min. Then 80μL 0.1mol / L ascorbic acid solution was added, and stirring continued vigorously at 1500rpm for 28s. Subsequently, 200μL Au seed solution was added to the above mixed solution, and stirring continued slowly at 600rpm for 50s, and then grown in a 25°C oven for 12h. After the reaction was completed, centrifugation and washing were performed, and the Au growth solution was obtained by concentration.

[0074] c. Preparation of Au@Ag nanorods:

[0075] 3mL 0.08mol / L CTAC (hexadecyltrimethylammonium chloride) was mixed with 0.2mL 10mmol / L silver nitrate, incubated at 25°C for 12min, and then 8mL Au growth solution was added to the mixed solution. After mixing, the mixture was incubated at 25°C for 6min, and then shaken at 28°C for 5h to obtain a brown-yellow product; centrifuged at 6000rpm for 17min to wash away excess CTAC; centrifuged again at 4500rpm for 17min to wash away unwrapped Au nanorods, and repeated 4 times, and the product was resuspended in 1mL ultrapure water to obtain Au@Ag nanorods.

[0076] S2. Preparation of flexible substrate

[0077] Food-grade silicone rubber A and curing agent B were uniformly mixed in a weight ratio of 1:1, and then cured at 90° C. for 5 minutes to obtain a flexible substrate.

[0078] S3. Assembly of flexible SERS substrate

[0079] a. Au@Ag nanorods self-assembly:

[0080] 5mL of Au@Ag nanorods were placed in a beaker, and then 1mL of n-hexane was added to it to quickly form an oil-water interface; then 2mL of ethanol was quickly injected into the system with a syringe, and an Au@Ag nanorod array was quickly formed at the oil-water interface. When the upper organic solvent evaporated at room temperature, the self-assembly of the Au@Ag nanorods was completed.

[0081] b. Assembly of flexible substrate: Use tweezers to hold a corner of the flexible substrate, gently cover it on the Au@Ag nanorod array, and quickly pick it up. The Au@Ag nanorod array will be directly and firmly printed on the surface of the flexible substrate. After drying under natural conditions, a self-assembled flexible SERS substrate can be obtained.

[0082] Example 3

[0083] The present application discloses a method for preparing a self-assembled flexible SERS substrate, comprising the following steps:

[0084] S1. Preparation of Au@Ag nanorods

[0085] a. Prepare Au seed solution: Use ultrapure water to prepare 0.2 mol / L CTAB solution, 0.1 mol / L tetrachloroauric acid solution, and use ice water to prepare 10 mmol / L sodium borohydride solution.

[0086] In a 32°C water bath, 2.5 mL of 0.2 mol / L CTAB (hexadecyltrimethylammonium bromide) solution was mixed with 2.5 mL of 0.1 mol / L tetrachloroauric acid solution, and gently stirred at 500 rpm for 2 min. A golden solution was formed after uniform stirring. Then, sodium borohydride solution was quickly added, and the solution changed from yellow to brown. At this time, it was vigorously stirred at 1500 rpm for 28 s, and then aged in a 32°C oven for 2-3 h, and stored in a 5°C refrigerator for use.

[0087] b. Preparation of Au growth solution: 1.4 g CTAB and 0.1234 g sodium oleate were stirred and dissolved in 25 mL ultrapure water at 55 ° C. After the temperature dropped to 25 ° C, 1.0 mL 10 mmol / L silver nitrate solution was added, and the mixture was stirred at 650 rpm for 5 min, and then allowed to stand in a 28 ° C oven for 18 minutes; then 25 mL 1 mmol / L tetrachloroauric acid solution was added, and stirring was continued at 650 rpm for 30-90 min until the solution turned milky white. At this time, 3 mL 1m HCl was added to adjust the pH of the solution to 5, and stirring was continued at 650 rpm for 10 min; then 85 μL 0.1 mol / L ascorbic acid solution was added, and stirring was continued vigorously at 1300 rpm for 30 s, and then 100 μL of Au seed solution was added to the above mixed solution, and stirring was continued slowly at 650 rpm for 70 s, and then grown in a 28 ° C oven for 16 h; after the reaction was completed, centrifugation and washing were performed, and the Au growth solution was obtained by concentration.

[0088] c. Preparation of Au@Ag nanorods:

[0089] 0.6mL 0.08mol / L CTAC (hexadecyltrimethylammonium chloride) was mixed with 0.2mL 10mmol / L silver nitrate, incubated at 28°C for 8min, and then 2.5mL Au growth solution was added to the mixed solution. After mixing, the mixture was incubated at 28°C for 4min, and then shaken at 32°C for 3h to obtain a brown-yellow product; centrifuged at 8000rpm for 125min to wash away excess CTAC; centrifuged again at 4700rpm for 12min to wash away unwrapped Au nanorods, and repeated 5 times, and the product was resuspended in 1mL ultrapure water to obtain Au@Ag nanorods.

[0090] S2. Preparation of flexible substrate

[0091] Food grade silicone rubber A and curing agent B were uniformly mixed in a weight ratio of 1:1 and incubated at 70°C for 10 minutes to obtain a flexible substrate.

[0092] S3. Assembly of flexible SERS substrate

[0093] a. Au@Ag nanorods self-assembly:

[0094] 3mL of Au@Ag nanorods were placed in a beaker, and then 1mL of n-hexane was added to it to quickly form an oil-water interface; then 1.5mL of ethanol was quickly injected into the system with a syringe, and an Au@Ag nanorod array was quickly formed at the oil-water interface. When the upper organic solvent evaporated at room temperature, the self-assembly of the Au@Ag nanorods was completed.

[0095] b. Assembly of flexible substrate: Use tweezers to hold a corner of the flexible substrate, gently cover it on the Au@Ag nanorod array, and quickly pick it up. The Au@Ag nanorod array will be directly and firmly printed and attached to the surface of the flexible substrate. After drying under natural conditions, a self-assembled flexible SERS substrate can be obtained.

[0096] Example 4

[0097] The present application discloses a method for detecting the activity of a self-assembled flexible SERS substrate, comprising preparing a probe molecule such as 4-ATP at a concentration of 10 -4 -10 -12 mol / L solution was dropped on the surface of the flexible SERS substrate to compare and test its SERS activity. The spectrum of SERS activity detection is shown in the attached figure. Figure 5 The concentration is 10 6 The SERS spectrum of the SERS activity detection of the mol / L 4-ATP solution dropped on the flexible substrate shows that compared with the single 4-ATP solution, the 1580 cm -1 A strong characteristic peak of the probe molecule appears nearby. This is due to the plasma resonance caused by Ag on the surface of Au nanorods, which enhances the SERS activity of the material. It can be seen that the Au@Ag flexible substrate has excellent enhancement activity for the probe molecule.

[0098] Example 5

[0099] The present application discloses an application of a self-assembled flexible SERS substrate in pesticide residue detection, which includes washing fresh apples, strawberries, and mushrooms purchased from the market with ultrapure water and then drying them naturally, preparing pesticide molecules such as thiram at a concentration of 10 -3 -10 -9 mol / L solution, spray it on the surface of the above fruits and vegetables. After drying naturally, cover the flexible SERS substrate assembled in Implementation Case 1 on the surface of the fruits and vegetables. At this time, the flexible SERS substrate will naturally stick to the surface. Wait for 10 seconds, remove it and use a portable Raman spectrometer for detection. The detector excitation wavelength is 785nm and the laser power is 50mW. After obtaining the Raman spectrum, the characteristic peaks are analyzed.

[0100] like Figure 6 This is the Raman spectrum obtained by using the flexible SERS substrate to detect pesticide residues in strawberries. It can be seen from the figure that this method can detect 1ppm of thiram, which is far below the detection limit in the national standard. Therefore, the flexible substrate provides a convenient, efficient and economical detection method for the actual rapid detection of pesticide residues in fruits and vegetables.

[0101] Usage principle and advantages: The present invention first adopts the seed method to prepare Au nanorods, then wraps silver nanoparticles in the outer layer, obtains Au@Ag by the liquid-liquid interface self-assembly method, and then mixes A and B glues in a ratio of 1:1 and solidifies them into a transparent silica-based film, and prints the Au@Ag film on the transparent silica-based film to assemble it into a flexible SERS substrate. The present application utilizes a self-assembly method to coat the formed Au@Ag nanorod single-layer two-dimensional film on a silica gel substrate. The preparation process is simple and fast, the raw materials are inexpensive, and the obtained substrate has good uniformity and high stability. The flexible SERS substrate has a negligible SERS background signal, and the Au@Ag flexible substrate has excellent enhancement activity, which makes the detection sensitivity high. The flexible SERS substrate has good plasticity and excellent flexibility, and can be folded and wiped flexibly on fruits and vegetables. The food-grade silica gel used is non-toxic and harmless, has few Raman characteristic peaks, and has little interference. When used in combination with a portable Raman instrument, it is particularly suitable as a flexible substrate for the detection of pesticide residues on the surface of fruits and vegetables. It has a practical application prospect that is simple, convenient, and operable, and provides a new method and technology for on-site rapid detection of pesticide residues in fruits and vegetables.

[0102] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a self-assembled flexible SERS substrate, characterized in that: The following steps are involved: S1. Preparation of Au@Ag nanorods a. Prepare Au seed solution: mix hexadecyltrimethylammonium bromide (CTAB) solution and tetrachloroauric acid solution under heating conditions, stir evenly to form a golden solution; then add sodium borohydride solution prepared with ice water to form a dark brown solution, increase the stirring speed and continue stirring for 28-32 seconds, then age at 28-32°C for 2-3 hours, and store at 3-5°C for use; b. Prepare Au growth solution: Dissolve CTAB and sodium oleate in ultrapure water at 45-55°C with stirring, wait until the temperature drops to 25-30°C, add silver nitrate solution, stir evenly and let stand at 25-30°C for 15-20 minutes; then add tetrachloroauric acid solution and continue stirring until the solution turns milky white, then adjust the pH to 4-5 and continue stirring for 10-20 minutes; then add ascorbic acid solution, increase the stirring speed and continue stirring for 28-32 seconds, then add Au seed solution to the above mixed solution, reduce the stirring speed and continue stirring for 50-70 seconds, and then grow at 25-30°C for 12-16 hours; after the reaction is completed, centrifuge and wash, and concentrate to obtain Au growth solution; c. Preparation of Au@Ag nanorods: Mix hexadecyltrimethylammonium chloride (CTAC) and silver nitrate, incubate at 25-30°C for 8-12 minutes, take out and add Au growth solution to the mixed solution, incubate at 25-30°C for 4-6 minutes, and then shake and react at 28-32°C for 3-5 hours to obtain a brown-yellow product; The excess CTAC was removed by centrifugation and washing, and the unwrapped Au nanorods were removed by centrifugation and washing again. After repeating 3-5 times, the product was resuspended in ultrapure water to obtain Au@Ag nanorods. S2. Preparation of flexible substrate After the silica gel A and the curing agent B are uniformly mixed in a weight ratio of 1:1, the mixture is cured at 70-90°C for 5-10 minutes to obtain a flexible substrate; S3. Assembly of flexible SERS substrate a. Au@Ag nanorods self-assembly: Au@Ag nanorods are placed in a container and n-hexane is added to form an oil-water interface. Ethanol is then injected into the system, and a self-assembled Au@Ag nanorod film is formed at the oil-water interface. When the upper organic solvent evaporates at room temperature, the self-assembly of the Au@Ag nanorods is completed. b. Assembly of flexible substrate: Au@Ag nanorod film is directly printed from a container using a flexible substrate and then dried at room temperature to obtain a self-assembled flexible SERS substrate.

2. The method for preparing a self-assembled flexible SERS substrate according to claim 1, characterized in that: In step S1-a, the concentration of the CTAB solution is 0.1-0.2 mol / L, the concentration of the tetrachloroauric acid solution is 0.05-0.1 mol / L, and the molar ratio of CTAB to tetrachloroauric acid is 1:0.25-0.5; The CTAB solution and the tetrachloroauric acid solution are mixed in a water bath, and the water bath heating temperature is 28-32°C; The stirring speed of the CTAB solution and the tetrachloroauric acid solution is 500-700 rpm, and the stirring speed after adding sodium borohydride is 1200-1500 rpm.

3. The method for preparing a self-assembled flexible SERS substrate according to claim 1, characterized in that: In the step S1-b, the mass ratio of CTAB to sodium oleate is 1:0.1-0.35; The molar ratio of silver nitrate, tetrachloroauric acid and ascorbic acid is 1:1.6-3.3:0.0053-0.008; The volume ratio of Au seed solution to the mixed solution is 1:400-550; The stirring speed after adding silver nitrate, tetrachloroauric acid, adjusting pH, and adding Au seed solution was 600-700 rpm; the stirring speed after adding ascorbic acid was 1200-1500 rpm.

4. The method for preparing a self-assembled flexible SERS substrate according to claim 1, characterized in that: In step S1-c, the molar ratio of silver nitrate to CTAC is 1:24-120; The volume ratio of Au growth solution to mixed solution is 1:0.32-0.4; The first centrifugation has a rotation speed of 6000-8000 rpm and a time of 12-17 min; the second centrifugation has a rotation speed of 4500-4700 rpm and a time of 12-17 min.

5. The method for preparing a self-assembled flexible SERS substrate according to claim 1, characterized in that: In step S2, the silica gel A is food grade silica gel.

6. The method for preparing a self-assembled flexible SERS substrate according to claim 1, characterized in that: In the step S3-a, the volume ratio of n-hexane, Au@Ag nanorods and ethanol is 1:3-5:1.5-2; the Au@Ag nanorod self-assembled film presents an Au@Ag nanorod array.

7. Use of a flexible SERS substrate prepared by the method for preparing a self-assembled flexible SERS substrate as claimed in any one of claims 1 to 6 in pesticide residue detection.

8. The use according to claim 7, characterized in that: Cover the self-assembled flexible SERS substrate on the surface of fruits and vegetables, wait for 10 seconds, and then uncover it and use a Raman spectrometer to detect it.

9. The use according to claim 8, characterized in that: The Raman spectrometer is a portable Raman spectrometer, the detector excitation wavelength is 785nm, and the laser power is 50mW.

10. The use according to claim 8, characterized in that: The fruits and vegetables include apples, strawberries, and mushrooms, and the pesticide residues include thiram.

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