Method for detecting 6-BA by alkali liquor assisted SERS (Surface Enhanced Raman Scattering) substrate AgNPs (at) GS material
By using lye-assisted SERS substrate AgNPs@GS material, a quantitative relationship model was constructed and ammonia was used for detection, which solved the problems of signal instability and insufficient sensitivity in traditional SERS detection methods, and achieved high sensitivity detection for 6-BA residues.
Patent Information
- Application Number
- CN202510069430.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-16
- Publication Date
- 2025-05-06
AI Technical Summary
When the existing SERS detection method is used for 6-benzyl aminopurine detection, it has defects such as unstable signal, complex substrate design, susceptible to food matrix interference, poor stability, and insufficient sensitivity.
Using alkali-assisted SERS substrate AgNPs@GS material, the construction of quantitative relationship model and the use of ammonia water significantly improves detection sensitivity and reduces interference.
The detection sensitivity of 6-BA pesticide residues was significantly improved, reaching the detection limit of 10-11 mol/L, and the detection limit of 6-BA residues in bean sprouts reached 5×10-4 mg/kg, which was lower than the existing SERS method.
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Abstract
Description
Technical Field
[0001] The invention relates to a method for detecting 6-BA by using an alkali solution-assisted SERS substrate AgNPs@GS material, and belongs to the technical field of surface enhanced Raman spectroscopy. Background Art
[0002] Raman spectroscopy has received a lot of attention in recent years as a powerful analytical technique that can provide detailed molecular information without the need for extensive sample preparation. This technique uses the inelastic scattering of monochromatic light to reveal vibrational, rotational and other low-frequency modes in a system. Among its various applications, surface-enhanced Raman scattering has emerged as an effective, direct and highly sensitive method for detecting trace amounts of analytes. SERS significantly amplifies the Raman signal through the interaction between molecules and metal nanostructures, mainly through two mechanisms: electromagnetic enhancement and chemical enhancement. Electromagnetic enhancement occurs when the analyte is adsorbed on the rough metal surface, resulting in localized electromagnetic field amplification, while chemical enhancement involves charge transfer interactions between the analyte and the metal, further enhancing the signal.
[0003] Based on the principle of SERS detection, this technology has been widely used in food safety research, especially in the detection of pesticide and veterinary drug residues. Given the potential health risks of pesticide residues in food, this issue has attracted increasing attention. The ability to detect these residues at trace levels is crucial to ensure consumer health and comply with regulatory standards. However, the practical application of SERS in daily food testing is often challenging due to the complexity of SERS substrate design and the need for various complex auxiliary methods to meet food safety standards.
[0004] In traditional surface enhanced Raman scattering (SERS) detection, commonly used substrate materials such as gold nanoparticles and silver nanoparticles, although they can enhance the Raman signal to a certain extent, still have defects such as low detection sensitivity, susceptibility to interference, and poor stability.
[0005] The existing detection methods for 6-benzylaminopurine mainly include high performance liquid chromatography (HPLC), gas chromatography (GC) and enzyme-linked immunosorbent assay (ELISA). These methods have the disadvantages of complex operation, insufficient detection sensitivity and high cost. When using traditional gold sol for Raman detection of 6-benzylaminopurine, although the detection process is simplified and the advantages of Raman detection are combined, it still faces the disadvantages of limited enhancement effect, unstable signal and matrix interference. Summary of the invention
[0006] [Technical issues]
[0007] When the existing traditional SERS detection method is used for 6-benzylaminopurine detection, it still has defects such as unstable SERS signal, complex substrate design, susceptibility to interference from food matrix, poor stability, and insufficient sensitivity.
[0008] [Technical solution]
[0009] In view of the defects of the prior art, the purpose of the present invention is to provide a method for detecting 6-BA using an alkali solution-assisted SERS substrate AgNPs@GS material. This method can effectively solve the problems of traditional SERS substrates in sensitivity, signal stability and interference suppression, significantly improve the detection sensitivity of 6-BA pesticide residues, and meet the strict requirements of food safety testing.
[0010] In order to achieve the above purpose, the technical solutions provided are as follows:
[0011] The present invention provides a method for detecting 6-benzylaminopurine (6-BA) using an alkali solution-assisted SERS substrate AgNPs@GS material, the method comprising the following steps:
[0012] (1) Construction of quantitative relationship model
[0013] A series of 6-BA standard solutions of the matrix were prepared, and then the 6-BA standard solutions were mixed with alkaline solution to obtain the test solution; the test solution was then dropped onto the AgNPs@GS material, and after it was completely dried, SERS detection was performed; the 1003±1cm -1 A quantitative relationship model was established between the Raman peak intensity at and the logarithm of 6-BA concentration;
[0014] (2) Determination of 6-BA content in the sample
[0015] The sample to be tested is mixed with the alkaline solution, and then the mixed solution is dripped onto the AgNPs@GS material. After it is completely dried, SERS detection is performed, and the 6-BA content in the sample to be tested is calculated based on the quantitative relationship model constructed in step (1).
[0016] In one embodiment, the alkali solution in step (1) is an aqueous solution of any one or more of ammonia, sodium hydroxide and lithium hydroxide; preferably an aqueous ammonia solution.
[0017] In one embodiment, the concentration of the alkaline solution in the test solution in step (1) is 0.01 to 10 mmol / L, preferably 10 mmol / L.
[0018] In one embodiment, the volume ratio of the 6-BA standard solution and the alkali solution mixed in step (1) is 1:1.
[0019] In one embodiment, the AgNPs@GS material in step (1) refers to a glass surface loaded silver nanoparticle SERS substrate material.
[0020] In one embodiment, the preparation of the AgNPs@GS material in step (1) comprises the following steps:
[0021] 1) A blank glass sheet was ultrasonically cleaned in ethanol, and then immersed in aqua regia; then rinsed with ultrapure water and dried for later use;
[0022] 2) mixing silver nitrate with cetylpyridinium chloride monohydrate solution (CPC) and stirring with a magnetic stirrer to form a milky white liquid;
[0023] 3) adding the dried glass piece from step 1) into the milky white liquid from step 2) and adding sodium hydroxide, the color quickly changes from milky white to brown; then placing it in a water bath at 35-40° C. for light-proof synthesis for 12-30 hours;
[0024] 4) After taking out the product after the reaction in step 3), rinse it with ultrapure water, and blow dry it under nitrogen to obtain AgNPs@GS material.
[0025] In one embodiment, the molar ratio of AgNO3, CPC and NaOH is 3:1:(40-160).
[0026] In one embodiment, the concentration of sodium hydroxide in step 3) is 0.5-2.0 mol / L; preferably 1.0 mol / L.
[0027] In one embodiment, the light-shielded synthesis time in step 3) is 24 hours.
[0028] In one embodiment, the parameters of the SERS detection in step (1) are: using a micro Raman imaging spectrometer (DXR2xi Raman Imaging Microscope) to measure the Raman spectrum of the sample addition area, the excitation wavelength of the portable Raman spectrometer is 785nm, the laser power is 10-30mW; the integration time is 0.01-1s.
[0029] In one embodiment, the test liquid in step (1) is added onto the AgNPs@GS material in small amounts and multiple times; wherein the amount added each time is 1 to 2 μL, and after each addition, the next addition operation is performed when the residual sample of the test sample is about to dry to form a smaller coffee ring; until the cumulative amount added reaches the detection amount.
[0030] In one embodiment, the sample to be tested in step (2) is an extraction solution of any one or more of bean sprouts, celery, rice, and apple.
[0031] The present invention also provides an application of the above-mentioned method in analyzing and detecting pesticides, veterinary drugs, dyes, food additives or prohibited food additives.
[0032] Beneficial effects:
[0033] The present invention provides a method for detecting 6-BA using an alkali solution-assisted SERS substrate AgNPs@GS material:
[0034] (1) On the one hand, it solves the defects of traditional detection methods for 6-BA residues, such as high cost, complicated operation, and poor stability, which cannot meet the people's growing demand for food safety; on the other hand, it solves the problems of poor stability and insufficient sensitivity when using traditional SERS to detect 6-BA residues;
[0035] (2) The present invention synthesizes a hydrophobic surface-enhanced Raman substrate with special stability and uniformity by a simple, rapid and low-cost method; by adding ammonia water to the test solution, the target molecule can be efficiently and rapidly enriched, and the SERS signal can be greatly enhanced. The detection method has a very high SERS detection sensitivity: using R6G as a Raman probe molecule can reach 10 -11 mol / L, and the detection limit of 6-BA residue in bean sprouts can reach 5×10 -4 The detection limit is 0.05-0.003 mg / kg, which is significantly lower than the existing SERS method (the detection limit of the existing technology for 6-BA residues in bean sprouts is between 0.05 and 0.003 mg / kg). BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 The graphs are obtained by SERS detection of R6G standard solutions with different concentrations in Example 2 of the present invention;
[0037] Figure 2 The SERS spectra and linear relationship fitting results of the ammonia-assisted AgNPs@GS material detecting different concentrations of 6-benzylaminopurine in Example 3 of the present invention; (a) is the SERS spectrum; (b) is the linear relationship fitting result diagram;
[0038] Figure 3 The SERS spectra and linear relationship fitting result diagram of the ammonia-assisted AgNPs@GS material detecting 6-benzylaminopurine residues in bean sprouts with different concentrations in Example 4 of the present invention; (a) is the SERS spectrum; (b) is the linear relationship fitting result diagram;
[0039] Figure 4The homogeneity results of AgNPs@GS materials measured in Example 5 of the present invention are shown in Figure 5; (a) Raman signal; (b) 1510 cm -1 The main vibration peak intensity distribution diagram at ;
[0040] Figure 5 This is a graph showing the stability results of the AgNPs@GS material measured in Example 5 of the present invention;
[0041] Figure 6 The SERS spectra of AgNPs@GS on the SERS substrate prepared under different NaOH concentrations and the 1510 cm -1 Raman peak intensity at 1510cm; (a) SERS spectrum; (b) 1510cm -1 The Raman peak is strong;
[0042] Figure 7 SERS spectra of AgNPs@GS prepared at different synthesis times on SERS substrate and 1510 cm -1 Raman peak intensity at 1510cm; (a) SERS spectrum; (b) 1510cm -1 The Raman peak is strong;
[0043] Figure 8 SEM images of the AgNPs@GS SERS substrates prepared at different synthesis times (a) 12 hours, (b) 18 hours, (c) 24 hours, (d) 30 hours; and the corresponding particle size distribution analysis results: (e) 12 hours, (f) 18 hours, (g) 24 hours, (h) 30 hours;
[0044] Fig. 9 The SERS spectra of different concentrations of ammonia assisted detection of 1 mg / kg 6-benzylaminopurine in Example 7;
[0045] Fig.10 The SERS spectra of different alkaline solutions assisted by AgNPs@GS in detecting 6-benzylaminopurine residues in bean sprouts in Example 8; (a) NaOH; (b) LiOH;
[0046] Fig.11 The SERS spectra of conventional gold sol in comparative example 1 for detecting different concentrations of 6-benzylaminopurine;
[0047] Fig.12 The SERS spectra and linear relationship fitting results of directly using AgNPs@GS to detect different concentrations of 6-benzylaminopurine in Comparative Example 2; (a) SERS spectrum; (b) linear relationship fitting results. DETAILED DESCRIPTION
[0048] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than 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 belong to the scope of protection of the present invention. The following specific implementation methods further describe the present invention.
[0049] The abbreviations of the names involved in the embodiments of the present invention are:
[0050] 6-BA: 6-benzylaminopurine;
[0051] AgNPs@GS: SERS substrate loaded with silver nanoparticles on glass surface.
[0052] Example 1
[0053] A method for preparing a glass surface loaded silver nanoparticle SERS substrate (AgNPs@GS) comprises the following steps:
[0054] (1) A blank glass sheet (15 mm × 40 mm) was ultrasonically cleaned three times in ethanol and then immersed in aqua regia for 24 hours; rinsed with ultrapure water and dried for later use;
[0055] (2) 10 mL of silver nitrate (45 mmol / L) was mixed with 10 mL of cetylpyridinium chloride monohydrate (15 mmol / L) solution and stirred with a magnetic stirrer for 30 min to form a milky white liquid;
[0056] (3) adding the dried glass piece from step (1) to the milky white liquid from step (2), and adding 4 mL of sodium hydroxide (1 mol / L), the color quickly changes from milky white to brown, and then placing it in a water bath at 35° C. for light-protected synthesis for 24 h;
[0057] (4) After the product of step (3) is taken out, it is rinsed with ultrapure water and blown dry under nitrogen to obtain a glass surface-loaded silver nanoparticle SERS substrate (AgNPs@GS).
[0058] Example 2
[0059] A method for testing the Raman enhancement performance of the glass surface-loaded silver nanoparticle SERS substrate prepared in Example 1 using rhodamine 6G as a Raman probe comprises the following steps:
[0060] (1) Dissolve R6G in ultrapure water to prepare 10 -1 mol / L R6G stock solution, and then diluted with ultrapure water to a series of R6G standard solutions with different concentrations;
[0061] (2) Add appropriate amounts of R6G standard solution of different concentrations to the surface of AgNPs@GS in small amounts and multiple times, wherein the amount added each time is 2 μL. After each addition, the next addition operation is performed until the sample to be tested is about to dry to form a small coffee ring; until the cumulative amount of R6G standard solution added reaches 10 μL; after the last addition is completed and the sample is completely dried, SERS detection is performed;
[0062] (3) The Raman spectrum of the sample addition area was measured using a Raman imaging microscope (DXR2xi Raman Imaging Microscope). The excitation wavelength of the portable Raman spectrometer was 785 nm and the maximum laser power was 30 mW. The measurement was performed using a laser power of 20 mW and an integration time of 0.25 s.
[0063] The results are as follows Figure 1 As shown, the analysis shows that 10 -11 The Raman spectrum of mol / L R6G solution still shows 613 cm -1 、774cm -1 、928cm -1 、1089cm -1 、1126cm -1 、1183cm -1 、1310cm -1 、1360cm -1 、1572cm -1 and 1651cm -1 The characteristic peak at , it can be seen that the detection limit of AgNPs@GS prepared in Example 1 for R6G is 10 -11 mol / L, indicating that the substrate has a good enhancement effect when used for SERS detection and can be used for SERS detection.
[0064] Example 3
[0065] A method for detecting 6-benzylaminopurine using a glass surface-loaded silver nanoparticle SERS substrate (AgNPs@GS) prepared in Example 1 with the assistance of ammonia, comprising the following steps:
[0066] (1) Mixing 6-BA standard solutions of different concentrations with 20 mmol / L ammonia solution at a volume ratio of 1:1 to obtain 6-BA-ammonia solution mixtures of different concentrations (wherein the ammonia concentration is 10 mmol / L);
[0067] (2) Adding appropriate amounts of 6-BA-ammonia water mixture of different concentrations to the surface of the AgNPs@GS substrate prepared in Example 1 in small amounts and multiple times, wherein the amount added each time is 2 μL. After each addition, the next addition operation is performed until the 6-BA-ammonia water mixture of the sample to be tested is about to dry to form a small coffee ring; until the cumulative amount of the 6-BA-ammonia water mixture added reaches 10 μL; after the last addition is completed and the sample is completely dried, SERS detection is performed;
[0068] (3) Using a Raman imaging microscope (DXR2xi Raman Imaging Microscope) to measure the Raman spectrum of the sample addition area, the excitation wavelength of the portable Raman spectrometer is 785 nm, the maximum laser power is 30 mW; the measurement is performed using a laser power of 20 mW and an integration time of 0.25 s;
[0069] (4) According to the Raman spectra of 6-BA-ammonia mixtures of different concentrations, the characteristic peak 1003 cm in the SERS spectrum of 6-BA-ammonia mixtures was selected. -1 A quantitative relationship was established between the Raman peak intensity at 6-BA and the logarithm of the 6-BA concentration in the 6-BA-ammonia water mixture, and a standard curve was drawn. The results are shown in Figure 2 (b)
[0070] like Figure 2 As shown in (a), the detection limit of 6-BA by ammonia-assisted glass surface-loaded silver nanoparticle SERS substrate (AgNPs@GS) is as low as 5×10 -5 mg / kg.
[0071] like Figure 2 As shown in (b), select 5×10 -5 A linear fit was performed in the range of 0.1 mg / kg to establish a linear relationship; the fitting equation of the standard curve drawn was I SERS =5270.71g(X C )+25367.8, when the actual sample concentration is 5×10 -5 There is a good linear relationship between 0.1 mg / kg and R 2 =0.99522.
[0072] Example 4
[0073] A method for detecting 6-benzylaminopurine (6-BA) in bean sprouts using a glass surface-loaded silver nanoparticle SERS substrate (AgNPs@GS) prepared in Example 1 with the assistance of ammonia, comprising the following steps:
[0074] (1) Preparation of bean sprout samples containing 6-BA residues:
[0075] Dissolve 6-BA in chromatographic grade methanol to prepare 6-BA stock solution, and then use ultrapure water to prepare a series of 6-BA standard solutions with different concentrations; soak 10g bean sprouts in 20mL of 6-BA standard solutions with different concentrations, let stand for 24 hours, take out and dry the water;
[0076] (2) taking 10 g of the dried bean sprouts from step (1), crushing them and placing them in a centrifuge tube, then adding 20 mL of methanol, performing ultrasonic extraction for 15 min, and then centrifuging at 5000 rpm for 10 min to collect the supernatant, repeating twice, and combining the supernatants;
[0077] (3) The collected combined supernatant was placed in a 50 mL chicken heart bottle and subjected to rotary evaporation at 40° C. and 60 rpm to remove methanol; the remaining residue was then rinsed with 10 mmol / L ammonia water and fixed to a final volume of 5 mL, thereby obtaining bean sprout sample solutions containing different concentrations of 6-BA to be tested;
[0078] (4) dripping the bean sprout sample solution containing different concentrations of 6-BA obtained in step (3) onto the surface of the AgNPs@GS prepared in Example 1 in small amounts and multiple times, wherein the dripping amount each time is 2 μL, and after each dripping is completed, the next dripping operation is performed until the 6-BA residual sample in the bean sprout sample to be tested is about to dry to form a small coffee ring; until the cumulative dripping amount reaches 10 μL; after the last dripping is completed and completely dried, SERS detection is performed;
[0079] (5) Using a Raman imaging microscope (DXR2xi Raman Imaging Microscope) to measure the Raman spectrum of the sample addition area, the excitation wavelength of the portable Raman spectrometer is 785 nm, the maximum laser power is 30 mW; the measurement is performed using a laser power of 20 mW, and the integration time is 0.25 s;
[0080] (6) According to the Raman spectrum of the 6-BA residual sample in bean sprouts, the characteristic peak 1003 cm in the SERS spectrum was selected -1 The quantitative relationship between the Raman peak intensity at 6-BA and the logarithm of the concentration of the bean sprout sample solution with different concentrations of 6-BA was established, and a standard curve was drawn. The results are as follows: Figure 3 (b) as shown.
[0081] like Figure 3 As shown in (a), the detection limit of 6-BA by ammonia-assisted glass surface-loaded silver nanoparticle SERS substrate (AgNPs@GS) is as low as 5×10 -4 mg / kg.
[0082] like Figure 3 As shown in (b), select 5×10 -4A linear fit was performed in the range of 1 mg / kg to establish a linear relationship; the fitting equation of the standard curve drawn was I SERS =2053.8lg(X C )+7965.5, when the actual sample concentration is 5×10 -4 There is a good linear relationship between 1mg / kg and R 2 =0.99316.
[0083] In order to verify the reliability of the ammonia-assisted glass surface-loaded silver nanoparticle SERS substrate (AgNPs@GS) in detecting 6-BA pesticide residues in bean sprouts, the pesticide residues in bean sprouts were determined by selecting the appropriate spike concentration based on the obtained standard curve. -4 Select any four concentrations in the range of 1 mg / kg to 1 mg / kg, namely 1 mg / kg, 0.1 mg / kg, 0.01 mg / kg, and 0.005 mg / kg.
[0084] The results are shown in Table 1. The measured recoveries were 100.98%, 90.27%, 106.40% and 91.40%, respectively. The RSD values were 4.59%, 2.19%, 4.24% and 5.91%, respectively. All of them met the requirements of GB / T 27404-2008 Laboratory Quality Control Specification for Physical and Chemical Testing of Food, indicating that this method can better detect 6-BA pesticide residues in bean sprouts and meet the detection needs.
[0085] Table 1. Recovery data
[0086]
[0087] Example 5
[0088] In order to further evaluate the uniformity and stability of AgNPs@GS prepared in Example 1, the following experimental investigations were conducted:
[0089] Add an equal amount of 10 -7 mol / L R6G standard solution, and randomly select 16 measurement points for detection according to the method of Example 2, and calculate their relative standard deviation (RSD) values.
[0090] The results are as follows Figure 4 As shown, R6G at 1510cm -1 The comparison of the main vibration peak intensity at shows that the relative standard deviation of the prepared SERS substrate is 8.45%, indicating that it has good uniformity.
[0091] The AgNPs@GS prepared in Example 1 was placed in a disposable plastic container and heated to 10 °C according to the method of Example 2. -7mol / L R6G standard solution was used to test the SERS effect at different time periods at room temperature.
[0092] The results are as follows Figure 5 As shown, it can be seen that even after storage at room temperature for 120 days, the SERS enhancement effect remains relatively stable, with only a 25.77% enhancement decrease. This result shows that the prepared AgNPs@GS substrate has significant stability and is suitable for ultra-sensitive target detection. The possible reason is that during the storage process, the outer layer of silver nanoparticles of the substrate are oxidized to form more stable silver oxide, thereby providing protection for the inner layer of silver nanoparticles, ensuring its long-term application capability in sensitive detection scenarios.
[0093] Example 6 Effect of different preparation conditions on the Raman enhancement performance of AgNPs@GS substrate
[0094] (1) is different from Example 1 only in that the concentrations of sodium hydroxide in step (3) are adjusted to 0.5 mol / L, 1.5 mol / L and 2.0 mol / L, respectively, and the other parameters and conditions are the same as those in Example 1.
[0095] The Raman enhancement performance of different AgNPs@GS prepared was tested according to the method of Example 2. The results are as follows: Figure 6 shown.
[0096] It can be seen that different sodium hydroxide concentrations have obvious effects on the Raman enhancement performance of the formed AgNPs@GS. The Raman enhancement performance of AgNPs@GS prepared at a NaOH concentration of 1 mol / L is better. Sodium hydroxide concentrations lower or higher than 1 mol / L will lead to poor substrate Raman enhancement performance, such as 0.5 mol / L, or 1.5-2 mol / L.
[0097] (2) The only difference from Example 1 is that the light-shielding synthesis time in step (3) is adjusted to 12 h, 18 h and 30 h respectively, and the other parameters and conditions are the same as those in Example 1.
[0098] The Raman enhancement performance of different AgNPs@GS prepared was tested according to the method of Example 2. The results are as follows: Figure 7 shown.
[0099] It can be seen that different synthesis times have obvious effects on the Raman enhancement performance of the formed AgNPs@GS. The Raman enhancement performance of the AgNPs@GS prepared at a synthesis time of 24 hours is better. Synthesis time that is too short (less than 12 hours) or too long (more than 30 hours) will lead to poor enhancement performance.
[0100] Furthermore, AgNPs@GS prepared with synthesis time of 12, 18, 24 and 30 hours were selected for scanning electron microscopy characterization, and the particle size distribution of silver nanoparticles in the electron microscope was analyzed.
[0101] Combination Figure 8 From the electron microscopy results, it can be seen that when the synthesis time of silver nanoparticles is 24 hours, the diameter of silver nanoparticles is relatively uniform, and the spacing meets the requirements of SERS hotspots. Image J was used for particle size analysis. At this time, the diameter of silver nanoparticles is about 40-60nm, and the spacing is about 1-10nm. During the period of 12-30 hours of silver nanoparticle synthesis, silver nanoparticles continue to grow on the glass surface, the particle size becomes larger, and the spacing becomes shorter. The most dense degree is reached when the deposition time is 24 hours. At this time, the diameter of silver nanoparticles is about 50nm, and the spacing is about 5nm. At this time, the particle size of silver nanoparticles is uniform and the spacing is close. After the deposition time is greater than 24 hours, silver nanoparticles continue to grow and gradually form aggregates with a size of about 70-200nm, and irregular silver nanoparticles such as rods are formed underneath. The results show that with the extension of growth time, the distribution of silver nanoparticles on the glass substrate transitions from sparse to dense, and the shape transitions from irregular to uniform, eventually leading to the appearance of irregular polymers. Under the optimal conditions, the diameter of the silver nanoparticles on the glass substrate is 40-60nm, showing a normal distribution. This shows that the glass substrate prepared under this condition is not only uniform in shape and regularly distributed, but also has a gap of about 5nm between individual silver nanoparticles, which provides a strong electromagnetic enhancement for the SERS signal.
[0102] Example 7 Effect of different concentrations of ammonia on the intensity of 6-BA detection signal
[0103] (1) 2 mg / kg of 6-BA was mixed with aqueous ammonia solutions of different concentrations (20, 2, 0.2, and 0.02 mmol / L) at a volume ratio of 1:1 to obtain mixed solutions of ammonia and 6-BA of different concentrations (the concentration of the pesticide in the solution was 1 mg / kg, and the concentration of ammonia was 10, 1, 0.1, and 0.01 mmol / L, respectively), which were the test solutions;
[0104] (2) dripping the test solution of step (1) onto the surface of the AgNPs@GS substrate prepared in Example 1 in small amounts and multiple times; wherein the amount of each dripping is 2 μL, and after each dripping is completed, the next dripping operation is performed until the test solution is about to dry to form a small coffee ring; until the cumulative dripping amount of the test solution reaches 10 μL; after the last dripping is completed and the substrate is completely dried, SERS detection is performed;
[0105] (3) The Raman spectrum of the sample addition area was measured using a Raman imaging microscope (DXR2xi Raman Imaging Microscope). The excitation wavelength of the portable Raman spectrometer was 785 nm and the maximum laser power was 30 mW. The measurement was performed using a laser power of 20 mW and an integration time of 0.25 s.
[0106] The results are as follows Fig. 9 As shown in the figure, after adding ammonia water, the SERS signal of 6-BA was significantly improved. With the increase of ammonia concentration, the SERS signal of 6-BA was gradually improved.
[0107] Example 8 Effects of different alkaline solutions on the SERS detection signal of 6-benzylaminopurine
[0108] (1) 6-BA standard solutions of different concentrations were mixed with 20 mmol / L NaOH solution and LiOH solution at a volume ratio of 1:1 to obtain mixed solutions of 6-BA of different concentrations and alkali solution of the same concentration (the concentration of 6-BA in the solution was 1, 0.1, 0.01, 1×10 -3 , ...mg / kg, the concentration of alkali solution is 10mmol / L), which is the test solution;
[0109] (2) dripping the test solution of step (1) onto the surface of the AgNPs@GS substrate prepared in Example 1 in small amounts and multiple times; wherein the amount of each dripping is 2 μL, and after each dripping is completed, the next dripping operation is performed until the test solution is about to dry to form a small coffee ring; until the cumulative dripping amount of the test solution reaches 10 μL; after the last dripping is completed and the substrate is completely dried, SERS detection is performed;
[0110] (3) The Raman spectrum of the sample addition area was measured using a Raman imaging microscope (DXR2xi Raman Imaging Microscope). The excitation wavelength of the portable Raman spectrometer was 785 nm and the maximum laser power was 30 mW. The measurement was performed using a laser power of 20 mW and an integration time of 0.25 s.
[0111] The results are as follows Fig.10 Shown by: Fig.10 As shown in (a) and (b), when NaOH and LiOH were used to assist in SERS detection of 6-BA, the detection limits were 1×10 -3 mg / kg and 5×10 -4 The detection limits are higher than those of ammonia-assisted 6-BA SERS detection.
[0112] This result shows that ammonia-assisted SERS detection of 6-BA is the best.
[0113] Comparative Example 1
[0114] A method for detecting 6-BA based on conventional gold sol Raman, the method comprising the following steps:
[0115] (1) Preparation of gold sol: 47 mL of ultrapure water and 3 mL of potassium chloroaurate solution (10 mg / mL) were added to a 100 mL round-bottom flask, and the solution was heated in an oil bath at 120° C. At the same time, a magnetic stirring device was turned on to stir at 565 r / min and the temperature was kept constant until the solution boiled; after the solution boiled, 2 mL of trisodium citrate aqueous solution (1% wt) was added and the solution was stirred at a constant temperature of 120° C. for 20 min until the solution turned purple-red. After the reaction was completed, the prepared gold sol was cooled to room temperature and stored in a refrigerator at 4° C. for later use;
[0116] (2) Wrap a glass slide with tin foil and lay it flat, drip the gold sol solution and the sample solution to be tested in sequence at the same position on it, and place it under a laser for detection; the laser light source wavelength of the portable Raman spectrometer is 785nm, the maximum laser power is 300mW, and the maximum scanning time is 25s.
[0117] The results are as follows Fig.11 As shown in the figure, it can be seen from the results that the detection limit of the gold sol synthesized by the traditional gold sol synthesis method and the traditional SERS detection method for the 6-BA standard solution is 0.05 mg / kg, which is much higher than the detection limit in Example 3.
[0118] Comparative Example 2
[0119] A method for detecting 6-benzylaminopurine (6-BA) by Raman analysis based on the AgNPs@GS substrate prepared in Example 1 comprises the following steps:
[0120] (1) Adding appropriate amounts of 6-BA standard solutions of different concentrations to the surface of the AgNPs@GS substrate prepared in Example 1 in small amounts and multiple times, wherein the amount added each time is 2 μL. After each addition, the next addition operation is performed until the sample to be tested is about to dry to form a small coffee ring; until the cumulative amount of the 6-BA standard solution added reaches 10 μL; after the last addition is completed and the sample is completely dried, SERS detection is performed;
[0121] (2) The Raman spectrum of the sample addition area was measured using a Raman imaging microscope (DXR2xi Raman Imaging Microscope). The excitation wavelength of the portable Raman spectrometer was 785 nm and the maximum laser power was 30 mW. The measurement was performed using a laser power of 20 mW and an integration time of 0.25 s.
[0122] (3) According to the Raman spectra of 6-BA standard solutions with different concentrations, the characteristic peak 1003 cm in the SERS spectrum of 6-BA was selected. -1 A quantitative relationship was established between the Raman peak intensity at and the logarithm of the concentration of the 6-BA standard solution, and a standard curve was drawn. The results are shown in Fig.12 (b) as shown.
[0123] like Fig.12 As shown in (a), the detection limit of 6-BA standard solution based on AgNPs@GS substrate is 0.01 mg / kg.
[0124] like Fig.12 As shown in (b), the range of 0.01 to 100 mg / kg was selected for linear fitting to establish a linear relationship; the fitting equation of the drawn standard curve is I SERS =7829.15g(X C )+19011.37, and has a good linear relationship between the actual sample concentration of 0.01 and 100 mg / kg, R 2 =0.99556.
[0125] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.
Claims
1. A method for detecting 6-BA using an alkali solution-assisted SERS substrate AgNPs@GS material, characterized in that: The method comprises the following: (1) Construction of quantitative relationship model A series of 6-BA standard solutions of the matrix were prepared, and then the 6-BA standard solutions were mixed with alkaline solution to obtain the test solution; the test solution was then dropped onto the AgNPs@GS material, and after it was completely dried, SERS detection was performed; the 1003±1cm -1 A quantitative relationship model was established between the Raman peak intensity at and the logarithm of 6-BA concentration; (2) Determination of 6-BA content in the sample The sample to be tested is mixed with the alkaline solution, and then the mixed solution is dripped onto the AgNPs@GS material. After it is completely dried, SERS detection is performed, and the 6-BA content in the sample to be tested is calculated based on the quantitative relationship model constructed in step (1).
2. The method according to claim 1, characterized in that The alkali solution in step (1) is an aqueous solution of any one or more of ammonia, sodium hydroxide and lithium hydroxide.
3. The method according to claim 1, characterized in that The concentration of the alkaline solution in the test solution in step (1) is 0.01-10 mmol / L.
4. The method according to claim 1, characterized in that: The volume ratio of the 6-BA standard solution and the alkali solution mixed in step (1) is 1:
1.
5. The method according to claim 1, characterized in that The AgNPs@GS material in step (1) refers to a SERS substrate material loaded with silver nanoparticles on the glass surface.
6. The method according to claim 1, characterized in that The preparation of the AgNPs@GS material in step (1) comprises the following steps: 1) A blank glass sheet was ultrasonically cleaned in ethanol, and then immersed in aqua regia; then rinsed with ultrapure water and dried for later use; 2) mixing silver nitrate with cetylpyridinium chloride monohydrate solution and stirring with a magnetic stirrer to form a milky white liquid; 3) adding the dried glass piece from step 1) into the milky white liquid from step 2), and adding sodium hydroxide, the color quickly changes from milky white to brown, and then placing it in a water bath at 35-40° C. for light-proof synthesis for 12-30 hours; 4) After taking out the product after the reaction in step 3), rinse it with ultrapure water, and blow dry it under nitrogen to obtain AgNPs@GS material.
7. The method according to claim 6, characterized in that In step 3), the concentration of sodium hydroxide is 0.5-2.0 mol / L.
8. The method according to claim 1, characterized in that: The parameters of the SERS detection in step (1) are: using a micro Raman imaging spectrometer to measure the Raman spectrum of the sample addition area, the excitation wavelength of the portable Raman spectrometer is 785nm, the laser power is 10-30mW; the integration time is 0.01-1s.
9. The method according to claim 1, characterized in that: In step (1), the liquid to be tested is dripped onto the AgNPs@GS material in small amounts and multiple times; wherein the amount of each dripping is 1 to 2 μL, and after each dripping is completed, the next dripping operation is performed until the residual sample of the sample to be tested is about to dry to form a smaller coffee ring; until the cumulative dripping amount reaches the detection amount.
10. Use of the method according to any one of claims 1 to 9 in analyzing and detecting pesticides, veterinary drugs, dyes, food additives or prohibited food additives.