Preparation method of nanomaterial with biomimetic enzyme activity and application of the nanomaterial in detection of glyphosate

Abstract

The application discloses a preparation method of nanometer material with biomimetic enzyme activity and application of the nanometer material in glyphosate detection, wherein the nanometer material with biomimetic enzyme activity is heptanoic acid and Prussian blue modified Fe3O4 nanoparticles, Fe3O4@C7 / PB with core-shell structure is obtained, the core is ferroferric oxide, and the shell layer is formed by compounding heptanoic acid and Prussian blue. The application has the beneficial effect that: the Fe3O4 nanoparticles modified by heptanoic acid and Prussian blue synthesize a nanometer enzyme (Fe3O4@C7 / PB) with peroxidase-like activity, the peroxidase-like activity is higher than that of Fe3O4@C7, and Fe3O4@C7 / PB can be kept stable through single-layer PB to prevent agglomeration. We establish an anti-interference intelligent mobile phone assisted nanometer sensing platform based on peroxidase-like activity of Fe3O4 nanoparticles (Fe3O4@C7 / PB) modified by heptanoic acid and Prussian blue (PB), which is used for determining glyphosate content in tobacco, and the detection limit is 0.1 ug / mL ‑1 .

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection technology, and in particular to a method for preparing nanomaterials with biomimetic enzyme activity and the application of such nanomaterials in the detection of glyphosate. Background Technology

[0002] Glyphosate is a highly effective, broad-spectrum, non-selective herbicide. Due to its high conductivity, cost-effectiveness, and systemic killing effect, it has been widely used in many fields, especially agriculture. Glyphosate has become one of the world's top herbicides, and its use continues to increase. Although the toxicity of glyphosate is currently controversial, large amounts of glyphosate residues can potentially cause poisoning to animals and humans through the food chain, and cases of poisoning have been reported. China, the US Environmental Protection Agency, and the European Union have all set limits on glyphosate residues. Glyphosate is a highly polar amino acid herbicide, insoluble in common organic solvents, lacks chromogenic and fluorescent groups, and has a strong binding ability to organic compounds in plants, making it difficult to analyze directly. To date, traditional analytical techniques for detecting glyphosate mainly include mass spectrometry, electrochemistry, ion chromatography, fluorescence spectroscopy, and various derivatization methods.

[0003] Nanomaterials with biomimetic enzyme activity (i.e., nanozymes) are typically mass-produced, low-cost, and more stable than natural enzymes, making them promising candidates for pesticide detection. A colorimetric sensing method based on nanozyme-catalyzed oxidation of chromogenic matrices and even trace targets has been reported. Most colorimetric nanozymes for pesticide detection are based on inhibiting nanozyme activity. Based on inhibiting the peroxidase-like catalytic activity of porous Co3O4 nanosheets, Luo et al. proposed a simple colorimetric nanozyme sheet for rapid detection of glyphosate in agricultural products. Liu et al. developed a system consisting of polyethyleneimine-coated upconversion nanoparticles, copper(II), hydrogen peroxide, and 3,3',5,5'-tetramethylbenzidine for the colorimetric and fluorescence determination of glyphosate. Yan et al. created a colorimetric determination of organophosphorus pesticides using peroxidase-inspired (POD) Fe3O4 nanoparticles. Since the first report of Fe3O4 nanoparticles in 2007, research has rapidly expanded to various nanomaterials, and Fe3O4 nanoparticles have also been modified with various functional groups to improve enzyme activity [17-19]. Similar to Fe3O4, Prussian blue (PB) is also composed of mixed-valence Fe with high POD-like activity. Therefore, colorimetric and fluorescence sensing platforms have great application prospects.

[0004] While current methods for detecting glyphosate are highly sensitive, sample preparation is more complex for complex samples, making these methods unsuitable for rapid, on-site detection. Furthermore, the catalytic activity of nanomaterials in complex samples is easily affected by surrounding environmental conditions. To address these issues, several analytical techniques have been developed, such as pretreatment techniques and smartphone-assisted sensing platforms. Smartphones, equipped with high-resolution cameras and capable of functioning as color cameras, have evolved into comprehensive tools for receiving, processing, and displaying biochemical or chemical substance detection data. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing nanomaterials with biomimetic enzyme activity and the application of such nanomaterials in glyphosate detection.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for preparing a nanomaterial with biomimetic enzyme activity, having a core-shell structure, involves modifying Fe3O4 nanoparticles with heptanoic acid and Prussian blue to obtain Fe3O4@C7 / PB with a core-shell structure, wherein the core is iron(III) oxide and the shell is composed of a composite of heptanoic acid and Prussian blue.

[0008] Preferably, in a N2 atmosphere, heptanoic acid, ammonia, and Prussian blue are added to a mixture of ferrous ammonium sulfate and ferric chloride to react and obtain a precipitate containing the biomimetic enzyme-active nanomaterial Fe3O4@C7 / PB.

[0009] Preferably, the reaction time is 0.5-2 hours and the reaction temperature is 60-90°C.

[0010] Preferably, the molar ratio of ferrous ammonium sulfate, ferric chloride, heptanoic acid, ammonia and Prussian blue in the reaction solution is 1:1-1.4:0.16-0.19:0.003-0.004:4-5.

[0011] As a preferred method, the precipitate is subjected to magnetic separation and washing to obtain Fe3O4@C7 / PB.

[0012] A method for detecting glyphosate using the aforementioned biomimetic enzyme-active nanomaterial includes the determination of glyphosate in a sample: adding the sample to be tested to a Fe3O4@C7 / PB+H2O2+ peroxidase matrix system, and quantitatively analyzing glyphosate by monitoring changes in system color or absorbance.

[0013] As a preferred option, standard curve plotting is also included:

[0014] Color images of the Fe3O4@C7 / PB+H2O2+ peroxidase matrix system under different glyphosate concentrations were captured, the RGB values ​​of the color images were obtained and converted into grayscale values, and a linear relationship between different glyphosate concentrations and grayscale values ​​was established.

[0015] Alternatively, the absorbance of the Fe3O4@C7 / PB+H2O2+ peroxidase matrix system at different glyphosate concentrations can be measured to establish a linear relationship between different glyphosate concentrations and absorbance.

[0016] Preferably, the sample pretreatment also includes extraction and decolorization. The decolorization step involves adding Al(OH)3 solution and NaOH solution to the extract, taking the supernatant, and performing glyphosate determination.

[0017] Preferably, the peroxidase substrate is 2,2′-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) or 3,3',5,5'-tetramethylbenzidine.

[0018] Preferably, the pH of the Fe3O4@C7 / PB+H2O2+peroxidase matrix system is 1.9-2.2, wherein the H2O2 concentration is 1.5-2.5 mM, the peroxidase matrix concentration is 0.15-0.25 mM, and the Fe3O4@C7 / PB concentration is 11-14 μg / mL. -1 .

[0019] Preferably, the samples include tea leaves, tobacco, soil, and environmental water samples.

[0020] The present invention has the following advantages:

[0021] A nanozyme (Fe3O4@C7 / PB) with peroxidase-like activity was synthesized using Fe3O4 nanoparticles modified with heptanoic acid and Prussian blue. Compared with Fe3O4@C7, it has higher peroxidase-like activity, and Fe3O4@C7 / PB can be stabilized by a monolayer of PB to prevent aggregation.

[0022] A potential problem in the determination of glyphosate content in tobacco products is false negative results caused by complex matrices. In this paper, we developed an interference-resistant smartphone-assisted nanosensor platform based on the peroxidase-like activity of heptanoic acid and Prussian blue (PB) modified Fe3O4 nanoparticles (Fe3O4@C7 / PB) for the determination of glyphosate content in tobacco, with a detection limit of 0.1 μg / mL. -1Three characteristic absorption peaks were observed at 416 nm, 647 nm, and 730 nm. Compared with Fe3O4, the newly synthesized Fe3O4@C7 / PB exhibited good peroxidase-like activity, which was evaluated in the presence of hydrogen peroxide using 2,2′-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a matrix. Furthermore, the catalytic activity of Fe3O4@C7 / PB was even inhibited by trace amounts of glyphosate.

[0023] Glyphosate molecules can occupy active sites on the surface of porous Fe3O4@C7 / PB nanoparticles, preventing the conversion of H2O2 to ·OH, thus causing a subtle color change in ABTS. By monitoring this color change, the concentration of glyphosate can be detected within 10 minutes. Based on this color change, and combining RGB color modeling and smartphone technology, a colorimetric quantitative method was developed for the quantification of glyphosate. This method is simple to operate, low in cost, and has a fast response time, showing great potential for field evaluation of glyphosate. Attached Figure Description

[0024] Figure 1 (A) TEM image of the prepared Fe3O4@C7. (B) TEM image of the prepared Fe3O4@C7 / PB. (C) XRD patterns of Fe3O4@C7 and Fe3O4@C7 / PB. (D) FTIR spectra of Fe3O4@C7 and Fe3O4@C7 / PB. (E) XPS spectra of Fe3O4@C7 and Fe3O4@C7 / PB. (F) Nuclear-level spectrum of Fe 2p in the Fe3O4@C7 / PB composite material.

[0025] Figure 2 (A) UV-Vis absorption spectra of the catalytic activity of Fe3O4@C7 / PB, TMB, and ABTS, and the inhibitory effect of glyphosate on different systems (B). (C) Catalytic activity of three different materials (Fe3O4, Fe3O4@C7, and Fe3O4@C7 / PB) on TMB. (D) UV absorption spectra of TMB oxidation in the presence of Fe3O4, Fe3O4@C7, and Fe3O4@C7 / PB.

[0026] Figure 3 Steady-state kinetic analysis of Fe3O4@C7 and Fe3O4@C7 / PB peroxidase-mimicking enzymes. Fe3O4@C7: (A) Rate-TMB concentration curve in the presence of H2O2; (C) Rate-H2O2 concentration curve in the presence of TMB; and (B,D) double reciprocal plots of (A,C). Fe3O4@C7 / PB: (E) Rate-TMB concentration curve in the presence of H2O2; (G) Rate-H2O2 concentration curve in the presence of TMB; and (F,H) double reciprocal plots of (A,C).

[0027] Figure 4 Fluorescence spectra of TA interacting with different systems (A); Elution effects of different eluents on glyphosate adsorbed on Fe3O4@C7 / PB (B). 1: Blank; 2: Water; 3: 1% NaOH aqueous solution; 4: Ethanol; 5: 1% NaOH ethanol solution; Raman spectra of different systems (C).

[0028] Figure 5 The purification effect of coprecipitation on tobacco samples (A); and the UV-Vis absorption spectra of tobacco samples before and after purification (B).

[0029] Figure 6 The colorimetric signal changes of the Fe3O4@C7 / PB+H2O2+ABTS system under different glyphosate concentrations are shown. Illustrations: Color changes of the nanozyme catalytic system (A); and the linear relationship between glyphosate concentration and absorbance (B).

[0030] Figure 7 Specificity of the colorimetric platform for glyphosate detection (A); interference from three phosphates (B); effects of IP, CaCl2, and IP+CaCl2 on the colorimetric platform (C); UV-Vis absorption spectra of the colorimetric platform when IP, CaCl2, and IP+CaCl2 are added, respectively (D). Illustration: Color changes of the colorimetric platform when IP, CaCl2, and IP+CaCl2 are added.

[0031] Figure 8 Linear relationship between grayscale values ​​read by smartphones and glyphosate concentration (A); and linear relationship between grayscale values ​​read by smartphones and glyphosate concentration (B).

[0032] Figure 9 A schematic diagram of a colorimetric sensor used for glyphosate detection. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0034] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0035] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0036] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0037] This application provides a method for preparing a core-shell structured nanomaterial with biomimetic enzyme activity. Fe3O4 nanoparticles are modified with heptanoic acid and Prussian blue to obtain Fe3O4@C7 / PB, where the core is iron(III) oxide and the shell is composed of heptanoic acid and Prussian blue. Specifically, in a N2 atmosphere, heptanoic acid, ammonia, and Prussian blue are added to a mixture of ferrous ammonium sulfate and ferric chloride to react and obtain a precipitate containing the biomimetic enzyme-active nanomaterial Fe3O4@C7 / PB. The precipitate is then magnetically separated and washed to obtain Fe3O4@C7 / PB. The reaction time is 0.5-2 h, and the reaction temperature is 60-90 °C.

[0038] This application also provides a method for detecting glyphosate using the aforementioned biomimetic enzyme-like nanomaterials, including standard curve plotting, sample pretreatment, and glyphosate determination in the sample.

[0039] Plotting the standard curve:

[0040] Color images of the Fe3O4@C7 / PB+H2O2+ peroxidase matrix system under different glyphosate concentrations were captured, the RGB values ​​of the color images were obtained and converted into grayscale values, and a linear relationship between different glyphosate concentrations and grayscale values ​​was established.

[0041] Alternatively, the absorbance of the Fe3O4@C7 / PB+H2O2+peroxidase matrix system at different glyphosate concentrations can be measured to establish a linear relationship between different glyphosate concentrations and absorbance.

[0042] Sample pretreatment:

[0043] The sample pretreatment includes extraction and decolorization. The decolorization step involves adding Al(OH)3 solution and NaOH solution to the extract, taking the supernatant, and determining glyphosate.

[0044] Determination of glyphosate in samples:

[0045] The sample to be tested was added to the Fe3O4@C7 / PB+H2O2+ peroxidase matrix system, and glyphosate was quantitatively analyzed by monitoring the color change or absorbance change of the system.

[0046] The peroxidase matrix can be 2,2′-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) or 3,3',5,5'-tetramethylbenzidine. The pH of the Fe3O4@C7 / PB+H2O2+peroxidase matrix system is 1.9-2.2, wherein the H2O2 concentration is 1.5-2.5 mM, the peroxidase matrix concentration is 0.15-0.25 mM, and the Fe3O4@C7 / PB concentration is 11-14 μg / mL. -1 The samples may include tea leaves, tobacco, soil, and environmental water samples.

[0047] Example:

[0048] 1. Materials

[0049] All reagents were commercially available analytical grade and, unless otherwise specified, were used as received. (NH4)2Fe(SO4)2·6H2O, heptanoic acid, Prussian blue (PB), FeCl3·6H2O, NH3·H2O (25% w / w), 2,2′-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), H2O2 (30% w / w), glyphosate, and other competitive organophosphorus pesticides (OPs) were supplied by Aladdin Reagents (Shanghai, China) Co., Ltd. Terephthalic acid (TA) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China). Deionized water for laboratory use was prepared using an ultrapure water system (18.23 MΩ·cm, UPT-II, Ulupure, China).

[0050] 2. Instruments

[0051] The morphology and microstructure of Fe3O4@C7 / PB were observed using a Tecnai G2 TF30 transmission electron microscope (TEM) with an accelerating voltage of 200 kV (FEI, USA). Ultraviolet-visible absorption spectra were measured using a TU-1901 double-beam ultraviolet-visible spectrophotometer manufactured by Beijing Purkinje General Analytical Instruments Co., Ltd. (Purkinje, China).

[0052] 3. Synthesis of Nanoparticles

[0053] Fe3O4@C7 / PB was prepared using a one-pot synthesis method. First, 3.38 g of (NH4)2Fe(SO4)2·6H2O and 2.82 g of FeCl3·6H2O were dispersed in 80 mL of deionized water. The suspension was heated to 80 °C under a nitrogen atmosphere with thorough stirring. Then, 200 mg of heptanoic acid (dissolved in 5 mL of acetone), 5 mL of NH3·H2O (28%, w / v), and 200 mg of Prussian blue (PB) were added to the solution stepwise. The mixture was stored at 80 °C for 1 h. After cooling the resulting precipitate to room temperature, it was magnetically separated and washed with deionized water and ethanol. The precipitate was lyophilized to obtain a powder. Finally, the obtained powder was redispersed in water, and the concentration of the nanomaterial was set to 3.24 mg / mL. -1 .

[0054] In this embodiment, the amounts of various reactants can fluctuate within a certain range. For example, the molar ratio of ferrous ammonium sulfate, ferric chloride, heptanoic acid, ammonia, and Prussian blue is 1:1-1.4:0.16-0.19:0.003-0.004:4-5. As long as Fe3O4@C7 / PB nanoparticles can be generated in the reaction solution, they can be separated by magnetic separation.

[0055] In this embodiment, considering the reaction rate and precipitation rate, the reaction temperature is set to 60-90℃. The precipitation rate is relatively fast within 0.5 hours of the reaction, and it can be completely precipitated in about 2 hours. However, considering the reaction efficiency, the optimal reaction time, i.e. precipitation time, is 1 hour.

[0056] 4. Kinetic tests of Fe3O4@C7 / PB

[0057] Under optimal conditions, the kinetics of the POD-like properties of Fe3O4@C7 / PB were investigated by varying the concentrations of TMB and H2O2. First, Fe3O4@C7 / PB (3.24 mg / mL) was used with a fixed H2O2 concentration (50 mM) and varying TMB concentrations (0.0625, 0.125, 0.1875, 0.25, 0.3125, 0.375, 0.4375, 0.5, 0.5625, 0.625 mM). -1 Analysis was conducted. Then, using H2O2 as the matrix, Fe3O4@C7 / PB / PB / PB / PB (3.24 mg / mL) with a fixed TMB concentration (0.25 mM) and variable H2O2 concentrations (0.625, 1.25, 1.875, 2.5, 3.125, 3.75) was used. -1 Experimental studies were conducted. The kinetic parameters were calculated based on the following Michaelis equations:

[0058] 1 / V = K m / Vmax ·1 / [s] +1 / V max

[0059] Where V is the initial velocity, V max [S] represents the maximum reaction rate, [S] represents the matrix concentration, and K represents the maximum reaction rate. m is the Michaelis constant. Using TMB and H2O2 as matrices, the KD of the POD-like activity of Fe3O4@C7 / PB was calculated. m Value and V max value.

[0060] 5. Peroxidase-like catalytic activity of Fe3O4@C7 / PB

[0061] The peroxidase-like activity of Fe3O4@C7 / PB was investigated by catalyzing several colorimetric reactions. Typically, 50 μL of 5 mM ABTS solution, 50 μL of (30%) H2O2 solution, and 50 μL of 1 mg / mL sodium hydroxide solution were added to 2.5 mL of 0.1 M NaAc-HAc buffer (pH 2.0). -1 Fe3O4@C7 / PB solution. UV-Vis spectra and absorbance values ​​were recorded at a fixed wavelength (416 nm) for analysis.

[0062] In this embodiment, the buffer solution is used to ensure the stability of the system and for color development. Those skilled in the art can choose other buffer solutions as needed. The amounts of each component in the system can also fluctuate within a certain range, but in order to ensure the sensitivity of glyphosate detection, the optimal amount was selected in this embodiment.

[0063] 6. Smartphone-based glyphosate detection

[0064] The detection experiment was conducted in an aqueous medium. First, 50 μL of 5 mM ABTS solution, 50 μL of (30%) H₂O₂ solution, and 50 μL of 1 mg / mL solution were added to the prepared sample. -1 Fe3O4@C7 / PB solution. Then, 0.1M NaAc-HAc buffer (pH 2.0) was added to adjust the volume of the resulting mixture to 2.5 mL. After the color change, an image was captured using a smartphone camera. Using a pre-installed color picker app, the obtained color images of different glyphosate concentrations were instantly converted into digital values ​​for red (R), green (G), and blue (B) channels for on-site quantitative analysis of glyphosate. Finally, the inhibitory effect was evaluated using inhibition efficiency (IE, %), and the glyphosate content was calculated. Inhibition efficiency (%) was calculated using the following formula: IE (%) = (A0 - A g ) / A0×100, where A gA0 and A0 represent the absorbance of the Fe3O4@C7 / PB-ABTS-H2O2 system at 416 nm in the presence and absence of glyphosate, respectively. The limit of detection (LOD) was determined using the 3σ criterion. All colorimetric glyphosate measurements were repeated in triplicate.

[0065] 7. Sample Pretreatment

[0066] For colorless liquid samples, the sample can be directly added to the Fe3O4@C7 / PB+H2O2+peroxidase matrix system. If the liquid sample is colored, it may affect the detection of glyphosate, so decolorization is required before adding it to the Fe3O4@C7 / PB+H2O2+peroxidase matrix system for determination. For solid samples, glyphosate needs to be extracted first. Those skilled in the art can select the sample pretreatment methods and approaches described in this application based on the sample conditions. This embodiment provides an optimal sample pretreatment method.

[0067] Taking a tobacco sample as an example, 1g of sample powder can be added to 30ml of deionized water (containing 1ml of sodium hydroxide (1M)). After sonication for 15 minutes, the resulting yellow solution is centrifuged at 8000rpm for 5 minutes. The supernatant is stored at 4℃ for subsequent decolorization experiments.

[0068] The extract was decolorized using a coprecipitation method. Simply put, 300 μL of Al(OH)₃ (0.33 M) was added to 2 mL of the extract. After mixing, 300 μL of NaOH (1 M) was added to the solution, and the mixture was vortexed for 30 seconds. Then, the mixture was centrifuged at 6000 rpm for 5 minutes. The upper glyphosate extract was used for enzyme inhibition analysis.

[0069] To avoid glyphosate contamination in the yellow precipitate, this study aimed to reduce glyphosate loss through secondary precipitation. 1 ml of deionized water was added to the yellow precipitate. After stirring for 1 min, the precipitate was dissolved in 300 μL of HCl (1M). Then, 300 μL of NaOH (1M) was added for secondary precipitation. After centrifugation, the supernatants from both extracts were combined and stored at 4°C for subsequent analysis.

[0070] 8. Results and Discussion

[0071] 8.1. Characteristic description of Fe3O4@C7 / PB

[0072] Figure 1TEM images A and B show the core-shell structures of Fe3O4@C7 and Fe3O4@C7 / PB. After PB modification, the particles tend to increase slightly in size. This is likely due to the PB modification on the Fe3O4@C7 surface. However, with increasing PB content, the particle size increases slightly, which may be related to the surface decomposition of Fe3O4@C7 during the reaction. The prepared Fe3O4@C7 / PB is stabilized by a monolayer of PB to prevent aggregation. During the preparation of Fe3O4@C7, heptanoic acid needs to be added slowly to slow down aggregation. In this embodiment of the application, PB can remain stable to prevent aggregation, therefore, the PB addition rate does not need to be controlled. The crystal structure of Fe3O4@C7 / PB was identified by XRD analysis. Figure 1 C). Diffraction peaks were observed at 2θ of 17.5°, 24.8°, 39.7°, and 51.0°, corresponding to diffraction planes at 200, 220, 400, and 440°, respectively. High-resolution XPS spectra of Fe 2p, C 1s (284 eV), and N 1s (401.2 eV) were fitted to Fe3O4@C7 / PB and the organic portion of the template (…). Figure 1 E). Fine Fe 2p XPS ( Figure 1 F) provides a good allocation to Fe 3+ 2p 1 / 2 Fe 2+ 2p 1 / 2 Fe 3+ 2p 3 / 2 and Fe 2+ 2p 3 / 2 The peaks were observed to verify the mixed valence state of Fe in the collected products. FTIR spectra of AuNPs / CDs and Fe3O4@C7 / PB were also examined. Figure 1 D) shows 2084cm -1 and 1412cm -1 The IR peak at point PB is in the γ(C≡N) stretching mode.

[0073] 8.2. Peroxidase-like activity of Fe3O4@C7 / PB

[0074] The catalytic performance of the prepared Fe3O4@C7 / PB on peroxidase substrates such as TMB and ABTS was evaluated. Figure 2 As shown in Figure A, when Fe3O4@C7 / PB reacts with H2O2 at room temperature, green (TMB) or steel blue (ABTS) colors can be observed. TMB (colorless → green) and ABTS (colorless → steel blue) show distinct color responses, with maximum absorption peaks at 650 nm and 730 nm, respectively. In acidic buffer solution, Fe3O4@C7 / PB exhibits the highest activity towards ABTS, approximately 5.0 times that of the TMB chromogen. Figure 2A). Due to the good affinity and sensitivity of Fe3O4@C7 / PB to ABTS, we chose ABTS as a colorimetric tool mimicking peroxidase activity for further smartphone-based quantitative analysis. In the absence of H2O2, the characteristic peaks at 650 nm and 730 nm disappeared. Figure 2 A) This indicates that Fe3O4@C7 / PB possesses peroxidase-like activity. For example... Figure 2 As shown in B, if SCN is applied before introducing Fe3O4@C7 / PB into the TMB / H2O2 solution... - Pretreatment will affect the POD activity of Fe3O4@C7 / PB by SCN. - The irreversible inhibition indicates that the Fe-Cx moiety is the main active site of POD. To further evaluate the peroxidase-like catalytic efficiency of Fe3O4@C7 / PB, the enzyme kinetic constant (Km) and maximum rate (Vmax) were obtained to measure the enzyme efficiency. Figure 3 When H2O2 and TMB were used as matrices, the Km values ​​of Fe3O4@C7 and Fe3O4@C7 / PB were 1.165 mM and 1.593 mM (H2O2 matrix), and 2.104 mM and 1.413 mM (TMB matrix), respectively, both significantly lower than that of HRP. This indicates that Fe3O4@C7 / PB has a higher affinity for the matrix than HRP. This may be due to the presence of more "active sites" on the Fe3O4@C7 / PB surface.

[0075] Table 1 Apparent Michaelis constants (K) for different NPs m ) and maximum reaction rate (V max )Compare

[0076]

[0077] 8.3. Effect of glyphosate on peroxidase-like activity of Fe3O4@C7 / PB

[0078] To investigate the effect of glyphosate on the peroxidase-like activity of Fe3O4@C7 / PB, the absorption spectra of different systems were measured. Figure 2 A and Figure 2 B). After the addition of glyphosate, the absorption peak at 730 nm in the Fe3O4@C7 / PB+H2O2+ABTS+glyphosate system was significantly reduced. No absorption peak was observed in either the ABTS+glyphosate or H2O2+ABTS+glyphosate systems, indicating that glyphosate cannot catalyze the oxidation of ABTS color change and can inhibit the peroxidase-like activity of Fe3O4@C7 / PB.

[0079] Therefore, the inhibition of Fe3O4@C7 / PB enzyme activity can be used for glyphosate detection.

[0080] 8.4. Mechanism of glyphosate inhibition of enzyme activity

[0081] Fe3O4@C7 / PB nanozymes can promote the generation of ·OH by decomposing H2O2, leading to the oxidation of the matrix ABTS. In the presence of glyphosate, the conversion of H2O2 to ·OH can be interrupted by occupying active sites on the Fe3O4@C7 / PB surface. To further investigate the inhibitory mechanism of glyphosate on the catalytic activity of Fe3O4@C7 / PB nanozymes, fluorescence experiments were used to track ·OH in the Fe3O4@C7 / PB+H2O2 system. Because terephthalic acid (TA) can be converted into 2-hydroxyterephthalic acid, a fluorescent agent with a peak at approximately 430 nm, TA was used to capture ·OH. Figure 4 As shown in Figure A, the fluorescence intensity of the Fe3O4@C7 / PB+H2O2+glyphosate system is lower than that of the Fe3O4@C7 / PB+H2O2 system, indicating that glyphosate can effectively inhibit the generation of ·OH. Furthermore, no fluorescence was observed when TA was cultured using Fe3O4@C7 / PB nanosheets, clearly indicating the absence of ·OH. The generated ·OH can also be directly detected by electron paramagnetic resonance (EPR) spectroscopy. Figure 4 B). The Fe3O4@C7 / PB+H2O2 system has a higher signal peak than the Fe3O4@C7 / PB+H2O2+glyphosate system, indicating that Fe3O4@C7 / PB has better catalytic activity.

[0082] These results confirm that the peroxidase activity of Fe3O4@C7 / PB can be inhibited by glyphosate.

[0083] When glyphosate is added to the Fe3O4@C7 / PB+ABTS+H2O2 system, the absorbance of the Fe3O4@C7 / PB+ABTS+H2O2 system decreases. Figure 2 B). When glyphosate adsorbed on Fe3O4@C7 / PB was eluted with different eluents (deionized water, 1% NaOH deionized water, ethanol, and 1% NaOH ethanol), the Fe3O4@C7 / PB-ABTS-H2O2 system eluted with 1% NaOH deionized water showed the best absorbance, indicating that the glyphosate adsorbed on Fe3O4@C7 / PB had been eluted. Figure 4B). This phenomenon confirms our hypothesis that the active site of Fe3O4@C7 / PB nanozyme is blocked by glyphosate. Surface-enhanced Raman spectroscopy (SERS) was used to reveal how the active site is blocked by glyphosate. Au NPs were synthesized according to the reported literature [HLaZZMingmingHan, Fast and Low-Cost Surface-Enhanced Raman Scattering (SERS) Method for On-Site Detection of Flumetsulam in Wheat, Molecules, 25(2020)4662]. Fe3O4@C7 / PB + glyphosate + Au NPs (437, 1344 cm⁻¹) -1 The SERS spectrum of the sample showed a much stronger Raman signal intensity than that of Au NPs and Fe3O4@C7 / PB-Au NPs, with values ​​at 797 and 905 cm⁻¹. -1 Two new signals were observed at ( Figure 3 C). From Figure 3 As can be seen from this, 437 and 797cm -1 The peak at this point is mainly formed based on the stretching vibrations of glyphosate molecules (Gaussian 09 procedure, density functional theory at the B3LYP / 6-31G(d) level). The results indicate that chemical bonds can form between the Fe3O4@C7 / PB surface and glyphosate.

[0084] 8.5. Purification and Method Optimization

[0085] The color of tobacco extracts significantly interferes with the results of colorimetric analysis. To improve the accuracy and stability of the proposed on-site testing sensing platform, a co-precipitation technique was used to pretreat the tobacco samples. Figure 5 As shown in Figure A, color interference was eliminated when Al(OH)3 and NaOH were added. To test whether glyphosate could be precipitated by coprecipitation, spiked water and tobacco samples were used to evaluate the coprecipitation purification technique. Glyphosate in the precipitate was also analyzed after being dissolved in 1 mL of HCl (1M). Table 2 shows that the precipitation efficiency of glyphosate was 1.87-3.23%, with relative standard deviations (RSD) ranging from 2.14-4.38%. This result indicates that the effect of coprecipitation on glyphosate detection is almost negligible. After purifying the sample using coprecipitation, the absorption peak at 250 nm in the tobacco sample was significantly reduced. Figure 5 B). At the same time, the background absorbance of the tobacco sample also decreased significantly, indicating that the co-precipitation technique can effectively eliminate background interference.

[0086] Table 2 Precipitation efficiency of glyphosate by coprecipitation method

[0087]

[0088] The parameters for optimizing the analytical performance of the Fe3O4@C7 / PB+ABTS+H2O2 system were optimized, including the concentration of Fe3O4@C7 / PB, pH, reaction time, and matrix concentrations of H2O2 and ABTS. Glyphosate inhibited the peroxidase-like activity of Fe3O4@C7 / PB because glyphosate molecules occupy active sites on the surface of porous Fe3O4@C7 / PB nanoparticles. Therefore, the concentration of Fe3O4@C7 / PB plays a crucial role in the color probe of the detection system. When the concentration of Fe3O4@C7 / PB is 12.5 μg / mL... -1 At different glyphosate concentrations, color differences in the system were easily discernible to the naked eye. Therefore, in subsequent experiments, the concentration of Fe3O4@C7 / PB was 12.5 μg / mL. -1 Then, to obtain optimal experimental results, the pH, reaction time, and matrix concentrations of ABTS and H2O2 were optimized. The reaction time was selected as 10 min, the pH value as 2, the H2O2 concentration as 2 mM, and the ABTS concentration as 0.2 mM for subsequent experiments.

[0089] 8.6. Performance of glyphosate determination

[0090] The peroxidase-like activity of Fe3O4@C7 / PB promotes the decomposition of H2O2 into hydroxyl radicals (·OH), which directly oxidizes ABTS to form a steel-blue product with three characteristic absorption peaks at 416 nm, 647 nm, and 730 nm. With increasing glyphosate concentration (…), Figure 6 A and Figure 6 B) The absorbance gradually decreased at 416 nm, 647 nm, and 730 nm, which was proportional to the concentration of glyphosate, indicating that the absorbance was within the range of 0.125–15 μg / mL. -1 The linear relationship was good within the range, with a correlation coefficient R greater than 0.99, and the detection limit was 0.1 μg / mL. -1 .

[0091] Specificity and anti-interference ability are important indicators for evaluating the detection capability of peroxidase-like nanozyme-based sensors. Other common pesticides (a-glyphosate, b-flufenamide, c-propargyl, d-thiamethoxam, e-atrazine, f-triphenyl phosphate, g-flubenzuron, h-dichlorophenoxyacetic acid, i-pentazocine, j-methyl parathion, k-cyhalothrin, and l-ziram) and phosphates (PO4) were selected. 3- HPO4 2- and H2PO 4- ) Evaluate the effect of interference. For example... Figure 7As shown, in the system, only glyphosate elicited a significant induced response, while other substances at the same concentration (10.0 mg / L) did not. -1 The pesticides had no obvious effect. Figure 7 A and Figure 7 B). However, phosphate (IP) causes a decrease in absorbance in the Fe3O4@C7 / PB-ABTS-H2O2 system, indicating that phosphate interferes with the detection system. To eliminate the interference of phosphate, we added calcium chloride (CaCl2), which reacts with the phosphate and CaCl2. 2+ Insoluble compounds are formed between them to eliminate interference. Figure 7 C and Figure 7 D).

[0092] 8.7. Smartphone Color Sensing Platform

[0093] Given the significance of on-site testing, portable devices and detection methods should also be considered. Based on the inhibitory effect of glyphosate on peroxidase activity, we designed a portable smartphone for on-site glyphosate detection, such as... Figure 9 As shown. The system was used to capture the colors of reaction solutions with different glyphosate concentrations, and further analysis was performed using a color recognition app installed on a smartphone, converting the color changes into RGB values. Since the brightness of the acquired probe solution images was negatively correlated with the glyphosate concentration, Adobe Photoshop CC 2015.5 software was used to read the grayscale values ​​from the smartphone, showing the correlation between different glyphosate concentrations (5-125 μg / mL). -1 Linear relationship between (R within the range) 2 =0.9973) Figure 8 As shown in A and 8B, the performance of the developed smartphone-assisted sensing platform is compared with other methods in the literature. As summarized in Table 3, the detection sensitivity of the proposed method is not as low as that of electrochemical sensors, but it has significant advantages in on-site detection and detection time.

[0094] Table 3 compares the glyphosate detection sensing platform with previously reported glyphosate detection platforms.

[0095]

[0096] 8.8. Determination of glyphosate content in actual samples

[0097] Considering the more complex matrix of tobacco samples compared to general agricultural products, different tobacco products were selected to evaluate the application of the smartphone colorimetric platform. As shown in Table 4, the average recovery rate of glyphosate in the actual spiked samples was 89.44-97.10%, with a relative standard deviation of 1.89-5.38%. Furthermore, the spiked recoveries obtained through the smartphone colorimetric platform were very similar to those obtained through GC-MS (Chinese National Standard GB / T 23750-2009). These results indicate that the smartphone colorimetric platform has good accuracy and repeatability, and has significant practical application value in the rapid detection of glyphosate in tobacco products.

[0098] Table 4. Determination of glyphosate in spiked tobacco samples (n=6).

[0099]

[0100] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting glyphosate, characterized in that, A method for preparing nanomaterials with biomimetic enzyme activity is disclosed: Fe3O4 nanoparticles are modified with heptanoic acid and Prussian blue to obtain Fe3O4@C7 / PB with a core-shell structure, wherein the core is iron(III) oxide and the shell is composed of heptanoic acid and Prussian blue; under a N2 atmosphere, heptanoic acid, ammonia, and Prussian blue are added to a mixture of ferrous ammonium sulfate and ferric chloride to react and obtain a precipitate containing the biomimetic enzyme-active nanomaterial Fe3O4@C7 / PB; the reaction time is 0.5-2 h and the reaction temperature is 60-90 °C; the molar ratio of ferrous ammonium sulfate, ferric chloride, heptanoic acid, ammonia, and Prussian blue in the reaction solution is 1:1-1.4:0.16-0.19:0.003-0.004:4-5; the precipitate is magnetically separated and washed to obtain Fe3O4@C7 / PB; The glyphosate detection method includes the determination of glyphosate in the sample: the sample to be tested is added to the Fe3O4@C7 / PB+H2O2+ peroxidase matrix system, and the glyphosate is quantitatively analyzed by monitoring the color change or absorbance change of the system. The peroxidase matrix is ​​2,2′-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) or 3,3',5,5'-tetramethylbenzidine; the pH of the Fe3O4@C7 / PB+H2O2+peroxidase matrix system is 1.9-2.2, wherein the H2O2 concentration is 1.5-2.5 mM, the peroxidase matrix concentration is 0.15-0.25 mM, and the Fe3O4@C7 / PB concentration is 11-14 μg / mL. -1 ; Color images of the Fe3O4@C7 / PB+H2O2+ peroxidase matrix system under different glyphosate concentrations were captured, the RGB values ​​of the color images were obtained and converted into grayscale values, and a linear relationship between different glyphosate concentrations and grayscale values ​​was established. Alternatively, the absorbance of the Fe3O4@C7 / PB+H2O2+peroxidase matrix system at different glyphosate concentrations can be measured to establish a linear relationship between different glyphosate concentrations and absorbance. The method also includes sample pretreatment, which includes extraction and decolorization. The decolorization step involves adding Al(OH)3 solution and NaOH solution to the extract, taking the supernatant, and then performing glyphosate determination.

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