Rapid and sensitive surface enhanced Raman scattering (SERS) detection method for glyphosate

Abstract

The invention discloses a rapid and sensitive SERS (Surface Enhanced Raman Scattering) detection method for GPH (Glyphosate Phosphate), which comprises the following steps: S1, preparing an SERS substrate, namely boiling AgNO3 and Na3-citrate solutions, and reacting until the solution is grey green to obtain an AgNPs solution; s2, performing a first-step derivatization reaction: adding a DMSO solution containing CS2 into 0.3-1.5 mL of a GPH solution, performing a reaction for 6-16 min, adding 10-30 [mu] L of 1 M NaOH, and performing a reaction for 3-6 min to obtain a reaction product A; s3, second-step derivatization reaction: adding the product A into an SERS (Surface Enhanced Raman Scattering) substrate, adding 120-180 [mu] L of 0.1 M HNO3, and uniformly mixing to obtain a reaction product B; and S4, performing SERS analysis: adding an aggregation inducer into the product B, and performing SERS analysis. According to the method disclosed by the invention, the amplification of the SERS signal of the final derivatization product is realized through the CS2-mediated two-step glyphosate derivatization reaction, so that the SERS signal intensity of the reaction system has GPH concentration dependence. The GPH detection time is shortened, the detection efficiency is greatly improved on the basis that high sensitivity of a traditional method is reserved, and the method is easy to operate and small in reagent consumption.

Description

Technical Field

[0001] This invention belongs to the field of glyphosate detection technology, specifically relating to a rapid and sensitive SERS detection method for glyphosate. Background Technology

[0002] Glyphosate (N-phosphonomethylglycine, GPH), the most widely used broad-spectrum herbicide globally, has led to persistent environmental residues and health risks due to its widespread application in modern agricultural production, particularly in glyphosate-resistant crop systems. Since the International Agency for Research on Cancer (IARC) classified GPH as a Group 2A possible human carcinogen in 2015, stringent maximum residue limits (MRLs) have been established globally; for example, the European Union sets the GPH residue limit in rice at 0.1 mg / kg. Therefore, there is an urgent need to develop reliable detection methods that combine high analytical performance with field applicability. While traditional techniques such as liquid chromatography-tandem mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) demonstrate excellent sensitivity and reliability in GPH monitoring, their practical application is limited. These methods typically require complex sample pretreatment, sophisticated instruments, and skilled operators, restricting their application in routine field monitoring. Immunoassays, while simple to operate, often struggle to distinguish GPH from its structural analogues and are costly. These technological and economic constraints have prompted researchers to develop new detection platforms that meet both regulatory sensitivity requirements and field applicability.

[0003] Surface-enhanced Raman spectroscopy (SERS) has become an important analytical technique in pesticide detection due to its molecular fingerprint specificity, rapid analytical capability, and compatibility with portable instruments. This technique utilizes the localized surface plasmon resonance (LSPR) effect of noble metal nanostructures to generate significant electromagnetic field enhancement. Common morphologies include spherical nanoparticles, anisotropic nanostructures, and hierarchical assemblies. However, SERS detection of small-molecule polar compounds such as GPH faces three major challenges: weak affinity between the analyte and the substrate, low intrinsic Raman scattering cross section, and matrix interference in complex food samples. Current mainstream solutions include: (1) chemical derivatization to enhance Raman activity; (2) nanostructure engineering to optimize electromagnetic hotspots; and (3) introducing molecular recognition elements. While theoretically effective, these methods have practical limitations: derivatization reaction conditions are often unsuitable for on-site detection, and recognition elements increase costs and reduce operational stability. Furthermore, the preparation of high-performance SERS substrates (through bottom-up or top-down methods) requires strict control of equipment parameters, etching conditions, and assembly environment. Therefore, the field of analytical chemistry urgently needs to develop simplified SERS methods that overcome these limitations while maintaining robustness.

[0004] Therefore, in order to address the above-mentioned technical problems, it is necessary to provide a rapid and sensitive SERS detection method for glyphosate.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid and sensitive SERS detection method for glyphosate, which can solve the problems mentioned in the background art.

[0007] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0008] A rapid and sensitive SERS detection method for glyphosate, characterized by comprising the following steps:

[0009] Preparation of S1 and SERS substrates:

[0010] The AgNPs solution was obtained by boiling the AgNO3 and Na3-citrate solutions until the solution turned grayish-green.

[0011] S2, First step of derivatization reaction:

[0012] Add 0.3-1.5 mL of GPH solution to DMSO solution containing CS2, react for 6-16 min, add 10-30 μL of 1 M NaOH, and react for another 3-6 min to obtain product A of the first derivatization reaction;

[0013] S3, Second step of derivatization reaction:

[0014] Product A was added to the SERS substrate, followed by 120-180 μL of 0.1 M HNO3. After mixing, product B from the second derivatization reaction was obtained.

[0015] S4 and SERS analysis:

[0016] SERS analysis was performed after adding an aggregation inducer to product B.

[0017] In one or more embodiments of the present invention, S1 specifically includes the following steps;

[0018] S11. Dissolve 10-30 mg AgNO3 in 60-90 mL of deionized water, place it in a three-necked round-bottom flask, and mix it with a magnetic stirrer.

[0019] S12. Heat the solution to boiling under vigorous stirring. After the temperature stabilizes, add Na3-citrate solution quickly 1-3 minutes under light-protected conditions.

[0020] S13. Continue boiling and stirring for 0.5-1.5 h until the mixture turns grayish-green, indicating that an AgNPs solution has been formed. After cooling to room temperature, this solution is used as a SERS substrate for subsequent experiments.

[0021] In one or more embodiments of the present invention, the AgNO3 in step S11 is 20 mg and the deionized water is 80 mL.

[0022] In one or more embodiments of the present invention, in step S12, 10 mL of 0.5%-2.5% (w / v) Na3-citrate solution is rapidly injected under light-protected conditions.

[0023] In one or more embodiments of the present invention, in step S2, the DMSO solution is 50 μL CS2 (4%, v / v) DMSO solution, and the NaOH is 20 μL 1 M; in step S3, the HNO3 is 150 μL 0.1 M; and in step S4, the aggregation inducer is 20 μL NaCl (10-100 mM).

[0024] In one or more embodiments of the present invention, the first step derivatization reaction product A is GPHCS2, and the second step derivatization reaction product B is ThzGPH.

[0025] In one or more embodiments of the present invention, step S4 specifically includes the following steps:

[0026] S41, GPH Quantitative Analysis;

[0027] After the second derivatization reaction product B has been in progress for 0.5-2 min, 15-30 μL of aggregation inducer is added. The mixture is used as the SERS test solution, and the SERS spectrum of the test solution is recorded for quantitative analysis of GPH.

[0028] S42. Establishing the standard curve;

[0029] GPH solutions of different concentrations were prepared by serial dilution. The SERS spectra of these solutions were measured using the S2-S4 method. A formula for determining the GPH concentration (C0) was established. GPH ) and 1214 cm -1 Linear relationship of SERS intensity (LgI) at the location:

[0030] LgI = k LgC GPH + b

[0031] Where k and b are the slope and intercept of the standard curve, respectively;

[0032] S43. Analysis of actual samples;

[0033] Rice was mixed with anhydrous ethanol and deionized water and thoroughly ground. The mixture was then centrifuged. The supernatant was filtered through a 0.22 μM membrane, and GPH was added to the filtrate to obtain a test solution containing GPH. SERS was then performed.

[0034] In one or more embodiments of the present invention, the aggregation inducing agent is 10-100 mM NaCl, and the SERS spectrum of the test solution is set with a laser power of 176.5 mW and an integration time of 3 s.

[0035] In one or more embodiments of the present invention, the concentration of the GPH solution in step S42 is 10. -3 -10 -7 M.

[0036] In one or more embodiments of the present invention, in step S43, 1-3 mL each of anhydrous ethanol and deionized water are mixed with 1-3 g of rice and thoroughly ground, and then centrifuged at 8000-12000 rpm for 6-16 min.

[0037] Compared with the prior art, the glyphosate rapid and sensitive SERS detection method of the present invention has the following advantages:

[0038] 1) The SERS signal of the final derivatized product was amplified through a two-step glyphosate derivatization reaction mediated by carbon disulfide, making the SERS signal intensity of the reaction system GPH concentration-dependent.

[0039] 2) The present invention improves the SERS signal of GPH derivatized products through the "adsorption-aggregation" mode. After 6 min of reaction, the signal is enhanced by 11.83% compared with the traditional "aggregation-adsorption" mode.

[0040] 3) Based on the controlled aggregation of AgNPs controlled by salt solution combined with the CS2-mediated two-step derivatization reaction of GPH, this invention achieves a GPH detection limit as low as 0.13 μM (S / N = 3) and achieves a recovery rate of 80.82%-129.62% in the spiked experiment on rice.

[0041] 4) It shortens the detection time of GPH, and while retaining the high sensitivity of traditional methods, it greatly improves the detection efficiency. In addition, the method is simple to operate and consumes little reagent, making it particularly suitable for rapid screening of GPH residues in agricultural and environmental samples. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0043] Figure 1 This is a schematic diagram of a rapid detection process for glyphosate (GPH) in one embodiment of the present invention;

[0044] Figure 2 This is a schematic diagram showing the characterization results of the SERS substrate using UV-Vis spectroscopy, Raman spectroscopy, TEM, and JmageJ software in Example 1.

[0045] Figure 3 This is a schematic diagram of the statistical results in Example 2;

[0046] Figure 4 This is a schematic diagram of the GPH SERS detection process in Example 3;

[0047] Figure 5 For example, LgI (1214 cm) in Example 3 -1 ) and LgC GPH A schematic diagram of the linear calibration curve;

[0048] Figure 6 In Example 3, at 10 -3 Schematic diagram of the ThzGPH SERS spectrum obtained from 40 repeated measurements at MgPH concentration;

[0049] Figure 7 For example, 1214 cm -1 Schematic diagram of SERS intensity distribution at the location;

[0050] Figure 8 To evaluate the anti-interference ability of the GPH SERS analysis method for amino acids in the application example;

[0051] Figure 9 The SERS detection and spiked recovery results of GPH (10 μM) in the actual sample in the application example. Detailed Implementation

[0052] To enable those skilled in the art to better understand the technical solutions in this disclosure, the technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this disclosure.

[0053] like Figure 1 As shown, where: GPH, glyphosate; GPHCS2, N-[(dithiocarboxyl)phosphonomethyl]glycine; ThzGPH, 5-oxo-(2-thiothiazolidin-3-yl)methyl)phosphonic acid.

[0054] A rapid and sensitive SERS detection method for glyphosate in one embodiment of the present invention includes the following steps;

[0055] Preparation of S1 and SERS substrates:

[0056] The AgNPs solution was obtained by boiling the AgNO3 and Na3-citrate solutions until the solution turned grayish-green.

[0057] S11. Dissolve 10-30 mg AgNO3 in 60-90 mL of deionized water, place it in a three-necked round-bottom flask, and mix it with a magnetic stirrer.

[0058] Preferably, 20 mg of AgNO3 is dissolved in 80 mL of deionized water and placed in a three-necked round-bottom flask, and then mixed on a magnetic stirrer.

[0059] S12. Heat the solution to boiling under vigorous stirring. After the temperature stabilizes, add Na3-citrate solution quickly 1-3 minutes under light-protected conditions.

[0060] Preferably, after the temperature stabilizes for 1 min, 10 mL of 0.5%-2.5% (w / v) Na3-citrate solution is rapidly injected under light-protected conditions.

[0061] S13. Continue boiling and stirring for 0.5-1.5 h until the mixture turns grayish-green, indicating that an AgNPs solution has been formed. After cooling to room temperature, this solution is used as a SERS substrate for subsequent experiments.

[0062] Preferably, the continuous boiling and stirring time is 1 hour.

[0063] S2, First step of derivatization reaction:

[0064] Add DMSO solution containing CS2 to 0.3-1.5 mL GPH solution, react for 6-16 min, add 10-30 μL 1 M NaOH, and react for another 3-6 min to obtain product A (GPHCS2) of the first derivatization reaction.

[0065] Preferably, 50 μL of CS2 (4%, v / v) DMSO solution is added to 1 mL of GPH solution, and the reaction is carried out at 25 °C for 10 min. Then, 20 μL of 1 M NaOH is added, and the reaction is carried out for 5 min to obtain product A of the first step of derivatization reaction.

[0066] S3, Second step of derivatization reaction:

[0067] Product A was added to the SERS substrate, followed by 120-180 μL of 0.1 M HNO3. After mixing, the product B (ThzGPH) from the second derivatization reaction was obtained.

[0068] Preferably, 10 μL of product A is added to 1 mL of the prepared AgNPs solution, and after rapid mixing, 150 μL of 0.1 M HNO3 is added immediately.

[0069] S4 and SERS analysis:

[0070] SERS analysis was performed after adding an aggregation inducer to product B.

[0071] S41, GPH Quantitative Analysis;

[0072] After the second derivatization reaction product B has been in progress for 0.5-2 min, 15-30 μL of aggregation inducer is added. The mixture is used as the SERS test solution, and the SERS spectrum of the test solution is recorded for quantitative GPH analysis. The aggregation inducer is NaCl (10-100 mM).

[0073] Preferably, after the second derivatization reaction product B has been in progress for 1 min, 20 μL of aggregation inducing agent NaCl (10-100 mM) is added, and the laser power for the SERS spectrum of the test solution is set to 176.5 mW and the integration time is 3 s.

[0074] S42. Establishing the standard curve;

[0075] Different concentrations of (10) were prepared by a stepwise dilution method. -3 -10 -7 M) GPH solution was prepared. SERS spectra of GPH solutions of different concentrations were measured using methods S2, S3, and S41. A formula was established based on the GPH concentration (C)... GPH ) and 1214 cm -1Linear relationship of SERS intensity (LgI) at the location:

[0076] LgI = k LgC GPH + b

[0077] Where k and b are the slope and intercept of the standard curve, respectively;

[0078] S43. Analysis of actual samples;

[0079] Mix 1-3 mL each of anhydrous ethanol and deionized water with 1-3 g of rice and grind thoroughly. Then centrifuge at 8000-12000 rpm for 6-16 min. Filter the supernatant through a 0.22 μM membrane and add GPH to the filtrate to obtain a test solution containing GPH. Then perform SERS test.

[0080] Preferably, 2 mL each of anhydrous ethanol and deionized water are mixed with 2 g of rice and thoroughly ground, then centrifuged at 10000 rpm for 10 min.

[0081] It should be noted that most of the materials and reagents used in this application were commercially available. Glyphosate (GPH, 99.5%) and glutamic acid (Glu, 99%) were purchased from Aladdin Industries, Inc. (Shanghai, China). Trisodium citrate (Na3-citrate) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Rhodamine 6G (R6G) and methionine (Met, 99%) were purchased from Shanghai Maclean Biochemical Co., Ltd. (Shanghai, China). Carbon disulfide was purchased from Shanghai Anaiji Chemical Co., Ltd. (Shanghai, China). Sodium chloride (NaCl) and sodium hydroxide (NaOH) were purchased from Guangdong Guanghua Science & Technology Co., Ltd. (Shantou, China). Glycine (Gly) was purchased from Beijing Bio-Top Technology Co., Ltd. Silver nitrate (AgNO3), dimethyl sulfoxide, nitric acid, and other chemical reagents were all purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Rice samples were purchased from a local supermarket (Nanjing, China).

[0082] The microstructure of the nanoparticles was characterized using high-resolution transmission electron microscopy (TEM, JEM-F200, NEC Corporation, Tokyo, Japan). Ultraviolet-visible (UV-Vis) absorption spectra were measured using a UV-1800 spectrophotometer (Shimadzu Corporation, Tokyo, Japan). Raman spectra were acquired using a portable Raman spectrometer (QE65pro, OceanOptics, USA) with an excitation wavelength of 785 nm. Other instruments used included: an ultrasonic cleaner (KQ-300DE, Kunshan Ultrasonic Instrument Co., Ltd., Kunshan, China), a magnetic stirrer (SZC, Tianjin Gongxing Laboratory Instrument Co., Ltd., Tianjin, China), a vortex oscillator (Cubic B, Guangzhou Siko Scientific Instrument Co., Ltd., Guangzhou, China), and a centrifuge (3K15, Sigma, Germany).

[0083] Example 1: Synthesis process and characterization of AgNPs;

[0084] 20 mg of AgNO3 was dissolved in 80 mL of deionized water and placed in a three-necked round-bottom flask. The solution was mixed thoroughly with a magnetic stirrer. The solution was heated to boiling with vigorous stirring. After the temperature stabilized for 1 min, 10 mL of 0.5%–2.5% (w / v) Na3-citrate solution was rapidly added under dark conditions. The mixture was boiled and stirred for 1 h until the mixture turned grayish-green, indicating the formation of the AgNPs solution. After cooling to room temperature, the AgNPs SERS substrate was obtained.

[0085] Figure 2 The results show the characterization of the SERS substrate using UV-Vis spectroscopy, Raman spectroscopy, TEM, and JmageJ software.

[0086] (a) Schematic diagram of UV-Vis spectra of AgNPs solutions prepared at different Na3-citrate concentrations;

[0087] (b) is a schematic diagram of the R6GSERS spectrum obtained through an AgNPs substrate;

[0088] (c) is 1505 cm -1 Signal strength diagram at the location;

[0089] (d) TEM image of AgNPs prepared with 0.5% Na3-citrate;

[0090] (e) TEM image of AgNPs prepared with 1.0% Na3-citrate;

[0091] (f) TEM image of AgNPs prepared with 2.0% Na3-citrate;

[0092] The insets in (d), (e), and (f) are the AgNPs particle size distribution diagrams under the current conditions.

[0093] from Figure 2 As can be seen from V-Vis characterization, with increasing Na3-citrate concentration, the surface plasmon resonance (SPR) peak of AgNPs exhibits a significant blue shift: the SPR peak of AgNPs synthesized with 0.5% sodium citrate is located at 442 nm, corresponding to an average particle size of 90.10 nm; while the SPR peak of AgNPs synthesized with 1.0% sodium citrate shifts to 418 nm, and the average particle size decreases to 79.05 nm. In SERS performance testing, with 10 -3 MR6G was used as a probe molecule for detection, and the results showed that AgNPs synthesized with 1.0% sodium citrate were at 1505 cm⁻¹. -1 The characteristic peaks exhibit the strongest signal response. Therefore, the AgNPs synthesized at a sodium citrate concentration of 1.0% possess excellent SERS activity, meeting the requirements for subsequent detection experiments.

[0094] Example 2: Optimization of salt-induced AgNP aggregation strategy;

[0095] First, 50 μL of CS2 (4%, v / v) DMSO solution was added to 1 mL of GPH test solution and reacted at 25 °C for 10 min. Then, 20 μL of 1 M NaOH was added, and the reaction was continued for 5 min to obtain derivatization product A (GPHCS2). Next, 10 μL of product A was added to 1 mL of prepared AgNPs solution, and after rapid mixing, 150 μL of 0.1 M HNO3 was immediately added and rapidly mixed again to obtain derivatization product B (ThzGPH). After reaction B had proceeded for 1 min, 20 μL of 70 mM aggregation inducing agent NaCl was added, and the mixture was used as the SERS test solution. The SERS spectrum of the test solution (laser power 176.5 mW; integration time 3 s) was recorded for quantitative GPH analysis.

[0096] like Figure 3 As shown,

[0097] (a) SERS spectral thermogram, where 1-10 represent 10-100 mM NaCl concentration.

[0098] (b) R6G based on AgNPs substrate at 1505 cm⁻¹ -1 SERS intensity at the location.

[0099] (c) is a schematic diagram of two aggregation modes ("aggregation before adsorption" and "adsorption before aggregation").

[0100] (d) is the 1214 cm⁻¹ of ThzGPH, a two-step derivatization product of GPH. -1 The change in the intensity of the characteristic peak over time.

[0101] (e) is a schematic diagram of the FDTD simulation results for monodisperse AgNPs;

[0102] (f) is a schematic diagram of the FDTD simulation results of aggregated AgNPs.

[0103] from Figure 3 It can be seen that the Raman intensity of R6G exhibits a clear concentration-dependent relationship with salt concentration, reaching its maximum enhancement effect at a NaCl concentration of 70 mM. (1505 cm⁻¹) -1 The characteristic Raman peak exhibits maximum intensity at 70 mM NaCl (i.e., Figure 3 (b) In this case, the nanoparticles reach their optimal aggregation state.

[0104] To elucidate the crucial impact of analyte adsorption sequence on the accessibility of the enhancement zone, this study used glyphosate two-step derivatization product (ThzGPH) as a model analyte and systematically compared two aggregation strategies. After the two-step derivatization reaction was completed, spectra were recorded every minute for 15 minutes to compare and analyze the two modes of "aggregation before adsorption" and "adsorption before aggregation" (e.g., ...). Figure 3 (Figures c-d in the table). In the pre-aggregation strategy, the ThzGPH signal intensity peaked at 6 min and then decreased. In contrast, the pre-adsorption strategy peaked at 1214 cm⁻¹. -1 The initial signal strength at the location (1 min) increased by 64.78%, and the peak strength at 6 min still maintained an increase of 11.83%. This enhancement is due to the optimized distribution of the analyte in the electromagnetic hotspot.

[0105] Finite-Domain Difference (FDTD) Simulation Figure 3 The results (ef) revealed the electromagnetic field distribution of monodisperse and aggregated AgNPs under 785 nm excitation. Compared with monodisperse nanoparticles, the gaps between AgNP aggregates showed a significantly enhanced electromagnetic field. These results indicate that precisely controlled salt-induced AgNP aggregation can effectively generate high-density electromagnetic hotspots. Therefore, optimal SERS response requires two necessary conditions: the formation of nanoscale gaps (hotspots) between plasmonic nanoparticles, and the maximal localization of reporter molecules in the electromagnetic field-enhanced region. These results strongly demonstrate that appropriate molecule pre-adsorption before inducing plasmonic nanoparticle aggregation can effectively improve SERS detection performance by optimizing molecule localization in electromagnetic hotspots.

[0106] Example 3: Performance Evaluation of GPH SERS Analysis

[0107] like Figure 4 As shown, the optimized GPH detection process is illustrated, with the entire process taking approximately 22 minutes. Figure 5 As shown, under optimized conditions, the characteristic peak is 1214 cm⁻¹. -1 The intensity of the signal (attributed to the ThzGPH thiazole ring vibration) exhibits a concentration-dependent response. Within the concentration range of 0.5–50 μM, the logarithm of the SERS signal intensity (LgI) correlates with the logarithm of the GPH concentration (LgC). GPH The regression equation showed a good linear relationship: LgI = 0.27 LgCGPH + 1.60 (R² = 0.911). The calculated limit of detection (LOD) for this method was 0.13 μM (signal-to-noise ratio S / N = 3).

[0108] like Figure 6 As shown, by analyzing 10 -3 The repeatability of the SERS detection system was systematically evaluated by performing 40 replicate measurements with MgPH standard solution. The 40 replicate SERS spectra showed good spectral quality. Figure 1 To the point of being consistent. For example... Figure 7 As shown, for 1214 cm -1 Quantitative analysis of the characteristic peaks showed that the relative standard deviation (RSD) of the signal repeatability was 8.39%, which fully meets the general standard of RSD < 20% in the validation of analytical methods.

[0109] Application example:

[0110] The matrix effect of amino acids in real samples can affect CS2-mediated GPH SERS detection; therefore, robustness to interference is crucial for method reliability. For example... Figure 8 As shown, using 10 -3 Met, Glu, and Gly of M were specifically validated as potential interferons, and the results showed that only GPH (10) -3 M) at 1214 cm -1 The generation of characteristic signals at the site demonstrates that this method has excellent selectivity for GPH detection and can effectively distinguish structurally similar amino acids.

[0111] like Figure 9 As shown, the GPH detection results in rice samples were calculated based on the standard curve plotted in Example 3, with spiked recoveries ranging from 80.82% to 129.62% (RSD < 20%).

[0112] Comparative Example 1

[0113] method Detection time LOD HPLC > 1 h <![CDATA[50 ng·mL –1 ]]> ELISA > 50 min <![CDATA[0.6 ng·mL –1 ]]> Colorimetric method > 32 min <![CDATA[28 ng·mL –1 ]]> SERS (This invention) 22 min <![CDATA[0.13 μM(~22 ng·mL –1 )]]>

[0114] Table 1. Schematic diagram comparing different glyphosate detection methods

[0115] In summary, compared with traditional methods (Table 1), this invention significantly shortens the GPH detection time (only 22 min), greatly improves the detection efficiency while retaining the high sensitivity of traditional methods, and the method is simple to operate with low reagent consumption, making it particularly suitable for rapid screening of GPH residues in agricultural and environmental samples.

[0116] This research addresses these challenges through an innovative combination of molecular engineering and nanomaterials science. For example... Figure 1 As shown, this method employs a two-step derivatization process, converting GPH to (5-oxo-(2-thiothiazolidin-3-yl)methyl)phosphonic acid (ThzGPH) via a CS2 reaction, while simultaneously introducing a strong Raman reporter group (with a 1214 cm⁻¹) -1 The thiazole ring and the strongly covalent AgNP group (thiol group) are prominent characteristic peaks. This chemical amplification, combined with an optimized salt solution-induced "adsorption-aggregation" mode of AgNPs, generates strong electromagnetic hotspots while maintaining signal reproducibility. The ordered "adsorption-aggregation" strategy ensures optimal localization of the analyte in the enhanced region, overcoming the limitation of traditional methods ("aggregation-adsorption") where reporter molecules struggle to enter dense electromagnetic "hotspots".

[0117] This study established three basic principles for the detection of small molecule SERS:

[0118] (1) Pre-adsorption of analytes achieves optimal location of "hot spots";

[0119] (2) Molecular design should combine metal-binding groups and Raman-active groups;

[0120] (3) Controllable aggregation kinetics require a balance between signal enhancement and nanoparticle stability. These principles provide insights for developing rapid SERS detection methods for various agrochemicals, environmental pollutants, and food contaminants.

[0121] It will be apparent to those skilled in the art that this disclosure is not limited to the details of the exemplary embodiments described above, and that this disclosure can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of this disclosure is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within this disclosure. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0122] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A rapid and sensitive SERS detection method for glyphosate, characterized in that, Includes the following steps: Preparation of S1 and SERS substrates: The AgNPs solution was obtained by boiling the AgNO3 and Na3-citrate solutions until the solution turned grayish-green. S2, First step of derivatization reaction: Add 0.3-1.5 mL of GPH solution to DMSO solution containing CS2, react for 6-16 min, add 10-30 μL of 1 M NaOH, and react for another 3-6 min to obtain product A of the first derivatization reaction; S3, Second step of derivatization reaction: Product A was added to the SERS substrate, followed by 120-180 μL of 0.1 M HNO3. After mixing, product B from the second derivatization reaction was obtained. S4 and SERS analysis: SERS analysis was performed after adding an aggregation inducer to product B.

2. The rapid and sensitive SERS detection method for glyphosate according to claim 1, characterized in that, S1 specifically includes the following steps; S11. Dissolve 10-30 mg AgNO3 in 60-90 mL of deionized water, place it in a three-necked round-bottom flask, and mix it with a magnetic stirrer. S12. Heat the solution to boiling under vigorous stirring. After the temperature stabilizes, add Na3-citrate solution quickly 1-3 minutes under light-protected conditions. S13. Continue boiling and stirring for 0.5-1.5 h until the mixture turns grayish-green, indicating that an AgNPs solution has been formed. After cooling to room temperature, this solution is used as a SERS substrate for subsequent experiments.

3. The rapid and sensitive SERS detection method for glyphosate according to claim 2, characterized in that, In step S11, the amount of AgNO3 is 20 mg and the amount of deionized water is 80 mL.

4. A rapid and sensitive SERS detection method for glyphosate according to claim 2 or 3, characterized in that, In step S12, rapidly inject 10 mL of 0.5%-2.5% (w / v) Na3-citrate solution under light-protected conditions.

5. The rapid and sensitive SERS detection method for glyphosate according to claim 3, characterized in that, In step S2, the DMSO solution is 50 μL of CS2 (4%, v / v) DMSO solution, and the NaOH is 20 μL of 1 M solution. In step S3, the HNO3 is 150 μL of 0.1 M solution, and in step S4, the aggregation inducer is 20 μL of NaCl (10-100 mM).

6. A rapid and sensitive SERS detection method for glyphosate according to claim 1 or 5, characterized in that, The first step derivatization reaction product A is GPHCS2, and the second step derivatization reaction product B is ThzGPH.

7. The rapid and sensitive SERS detection method for glyphosate according to claim 6, characterized in that, S4 specifically includes the following steps: S41, GPH Quantitative Analysis; After the second derivatization reaction product B has been in progress for 0.5-2 min, add 15-30 μL of aggregation inducer. The mixture is used as the SERS test solution, and the SERS spectrum of the test solution is recorded for quantitative analysis of GPH. S42. Establishing the standard curve; GPH solutions of different concentrations were prepared by serial dilution. The SERS spectra of these solutions were measured using the S2-S4 method. A formula for determining the GPH concentration (C0) was established. GPH ) and 1214 cm -1 Linear relationship of SERS intensity (LgI) at the location: LgI = k LgC GPH + b Where k and b are the slope and intercept of the standard curve, respectively; S43. Analysis of actual samples; Rice was mixed with anhydrous ethanol and deionized water and thoroughly ground. The mixture was then centrifuged. The supernatant was filtered through a 0.22 μM membrane, and GPH was added to the filtrate to obtain a test solution containing GPH. SERS was then performed.

8. The rapid and sensitive SERS detection method for glyphosate according to claim 7, characterized in that, The aggregation inducing agent is 10-100 mM NaCl, and the SERS spectrum of the test solution is set with a laser power of 176.5 mW and an integration time of 3 s.

9. The rapid and sensitive SERS detection method for glyphosate according to claim 8, characterized in that, The concentration of the GPH solution in step S42 is 10. -3 -10 -7 M.

10. A rapid and sensitive SERS detection method for glyphosate according to claim 9, characterized in that, In step S43, 1-3 mL each of anhydrous ethanol and deionized water are mixed with 1-3 g of rice and thoroughly ground, then centrifuged at 8000-12000 rpm for 6-16 min.

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