A method for glyphosate detection based on paper-based field amplification enrichment and fluorescence

Abstract

The present invention discloses a method for paper-based field amplification enrichment and fluorescence detection of glyphosate, belonging to the field of analytical detection technology. The present invention relates to a method for paper-based field amplification enrichment and fluorescence detection of glyphosate, comprising: (1) forming a fluorescence enrichment band: using a glass fiber membrane as a paper channel, dripping a sample containing glyphosate and a fluorescence developer into the sample loading area and fluorescence color development area of ​​the paper channel, respectively; and adding polyvinyl pyrrolidone to the cathode electrolyte cell; applying a constant voltage for field amplification enrichment, and obtaining a fluorescence-enhanced enrichment band in the fluorescence color development area; (2) determining the glyphosate content: using the grayscale ratio of the fluorescence-enhanced enrichment band to establish a linear relationship with the glyphosate concentration, obtaining a glyphosate standard curve; and calculating the glyphosate content in the glyphosate sample to be tested. This method can detect glyphosate in water and soil samples, is fast, easy to operate, highly sensitive, and has practical application prospects.

Description

Technical Field

[0001] The invention relates to a method for glyphosate based on paper-based field amplification enrichment and fluorescence detection, belonging to the technical field of analysis and detection. Background Art

[0002] Glyphosate is an organophosphorus herbicide widely used for weed control in agriculture due to its low production cost, high efficacy, broad spectrum, and excellent systemic transport in plants. The widespread use of glyphosate causes its migration into the environment. While some glyphosate is degraded through biological and photochemical pathways, some glyphosate and its metabolites remain in soil and rivers, accumulating in the food chain and posing potential hazards to humans. These include symptoms such as cardiac arrhythmias, hypotension, psychotic episodes, and renal and respiratory failure. Furthermore, there is still no definitive conclusion on the carcinogenicity of glyphosate. Therefore, the Food and Agriculture Organization of the United Nations (FAO) has established that glyphosate residues in food must be below the Dietary Daily Intake (ADI) and Maximum Residue Limits (MRLs). The MRLs for different food types range from 0.025 to 20 mg / kg. Some cereals, including rice, wheat, and oats, have an MRL of 20 mg / kg, while meat, legumes, and milk have MRLs of 0.05, 2, and 0.052 mg / kg, respectively. According to GB2763-2021, "National Food Safety Standard Maximum Residue Limits of Pesticides in Food," my country also has established limits for glyphosate residues in food, with MRLs ranging from 0.1 to 7 mg / kg for different types of food. Therefore, a rapid on-site detection method for glyphosate in food matrices is urgently needed.

[0003] Currently, the most commonly used method for glyphosate detection is chromatography, including high-performance liquid chromatography (HPLC), gas chromatography (GC), and ion chromatography (IC). Another method is liquid chromatography-mass spectrometry (LC-MS), where the eluate from the chromatographic column is fed into a mass spectrometer for analysis.

[0004] HPLC, as the most commonly used method for glyphosate detection, usually requires pre-column or post-column derivatization and is used in conjunction with ultraviolet / visible spectrophotometry (UV / Vis) or fluorescence (FL) detectors after derivatization. GC is usually used to detect glyphosate after pre-column derivatization to convert it into volatile and thermally stable derivatives. These methods all require a complex derivatization process and are time-consuming. IC uses two eluents with different pH values ​​and ionic strengths for gradient elution and does not require derivatization, but there are ion competition and other environmental changes such as pH, organic matter and microorganisms, which make extraction difficult and the results cannot be well reproduced. In addition, chromatography requires professional instruments and equipment and is relatively expensive. Compared with these methods, fluorescence analysis methods have the advantages of high efficiency, strong environmental adaptability, low cost, easy operation, and considerable reaction rate. Therefore, they are widely used in the specific detection of biologically active oxygen, various metal ions and some organic small molecules. However, most of the existing fluorescence detection methods for glyphosate require the introduction of some media to obtain the fluorescence response of glyphosate, such as the selection of Cu 2+ This step complicates the detection system and introduces Cu 2+ There is a possibility that quenchers such as these will bind to or interact with matrix substances, causing analytical errors.

[0005] Metal-organic frameworks (MOFs) are a class of crystalline materials composed of metal ions linked by organic bridging ligands, exhibiting ultrahigh porosity and a large internal surface area. Leveraging the inherent optical and photonic properties of the metal ions and organic ligands within their structural composition, as well as the collaborative assembly or encapsulation of guest species within their frameworks, a range of fluorescent MOFs have been developed as platforms for photofunctional materials. However, MOF-based fluorescent sensors still require specialized equipment such as fluorescence spectrometers, making them unsuitable for rapid on-site detection of glyphosate.

[0006] A paper-based analytical device (PAD) is a portable, on-site detection device based on paper. It offers advantages such as low cost, simple manufacturing, non-toxicity, and environmental friendliness. However, when fluorescence analysis is combined with PAD, sensitivity is limited, making accurate quantitative analysis impossible.

[0007] Online electrokinetic stacking technology can effectively achieve pre-concentration, thereby improving the sensitivity of PAD. Among them, field amplification stacking (FAS) is one of the most effective and simplest online pre-concentration methods, which achieves the enrichment of the target by adjusting the electric field strength and electroosmotic flow.

[0008] Here, we constructed a glasses-type paper-based field-amplified enrichment device (GFA-PAD) and synthesized a new fluorescent MOF material NH2-Bi-BDC via a hydrothermal method. We applied it to the GFA-PAD and combined it with ultraviolet imaging and data analysis to establish a method for paper-based field-amplified enrichment and fluorescence detection of glyphosate. This method was successfully applied to the online enrichment and fluorescence detection of glyphosate in water samples (lake water, tap water, drinking water) and soil. Summary of the Invention

[0009] To address these issues, the present invention constructed a glasses-type paper-based field-amplified enrichment device (GFA-PAD). A novel fluorescent MOF material, NH2-Bi-BDC, was synthesized via a hydrothermal method and applied to the GFA-PAD, providing a glasses-type paper-based field-amplified enrichment and fluorescence detection method for glyphosate. This method enables the rapid and convenient detection of glyphosate in food samples, demonstrating promising practical applications.

[0010] The amino-functionalized metal-organic framework itself has fluorescent properties. Because of the hydrogen bond interaction between the amino groups exposed on the surface and the carboxyl groups of glyphosate, it can promote excited-state proton transfer (ESPT), directly causing fluorescence signal enhancement, and can achieve rapid fluorescence detection of glyphosate.

[0011] The object of the present invention is to provide a method for the enrichment and fluorescence detection of glyphosate based on a paper-based field amplification method, the method comprising the following steps:

[0012] (1) Formation of fluorescent enrichment bands

[0013] A glass fiber membrane serves as a paper channel, and is combined with a DC high-voltage power supply, an electrolyte pool, and a platinum wire electrode to form a paper-based field amplification device. The platinum wire electrode is immersed in the cathode and anode electrolyte pools, respectively, and polyvinyl pyrrolidone is added to the cathode electrolyte pool. The paper channel is sequentially divided into a sample loading zone, a sample enrichment zone, and a fluorescence development zone. The fluorescence development zone is located near the anode, and the sample loading zone is located near the cathode. A sample containing glyphosate is dripped into the sample loading zone, and a fluorescence developer is dripped into the fluorescence development zone. A constant voltage is applied to cause glyphosate to migrate through the sample enrichment zone for enrichment. When the enriched glyphosate meets NH2-Bi-BDC, a reaction occurs, resulting in a fluorescence-enhanced enrichment band in the fluorescence development zone.

[0014] (2) Determination of glyphosate content

[0015] According to step (1), a series of standard samples of known concentrations and the fluorescence enrichment color bands of the glyphosate sample to be tested are obtained respectively, and image information is obtained by photographing and imaging under ultraviolet light, and grayscale analysis is performed. The grayscale ratio of the fluorescence enhanced band before and after the addition of glyphosate is used as the basis for glyphosate quantitative analysis, and a linear relationship between the grayscale ratio and the glyphosate concentration is constructed to obtain a glyphosate standard curve; at the same time, based on the standard curve, the glyphosate content in the glyphosate sample to be tested is calculated.

[0016] In one embodiment, the mass concentration of the polyvinyl pyrrolidone in step (1) relative to the cathode electrolyte is 0.5-2.0%.

[0017] In one embodiment, the mass concentration of the polyvinyl pyrrolidone in step (1) relative to the cathode electrolyte is 1.0%.

[0018] In one embodiment, the electrolyte in the electrolyte pool of step (1) is a pH 6.5 3-(N-morpholino)propanesulfonic acid buffer solution with a concentration of 25 to 75 mmol / L.

[0019] In one embodiment, the concentration of the electrolyte is 50 mmol / L.

[0020] In one embodiment, the loading amount of the glyphosate sample to be tested and the color developing fluorescent agent in step (1) is 10 to 20 μL.

[0021] In one embodiment, the concentration of the fluorescent developer in step (1) is 0.025 to 0.1 g / L.

[0022] In one embodiment, the concentration of the fluorescent developer in step (1) is 0.025 g / L.

[0023] In one embodiment, the constant voltage in step (1) is 200-220V.

[0024] In one embodiment, the enrichment time in step (1) is 60 to 160 seconds.

[0025] In one embodiment, the enrichment time in step (1) is 100 s.

[0026] In one embodiment, the ultraviolet lamp irradiation wavelength in step (2) is 365 nm.

[0027] In one embodiment, the grayscale analysis in step (2) is to perform grayscale analysis on the image obtained by taking a photo using Image J software.

[0028] In one embodiment, the fluorescent display agent in step (1) is a fluorescent MOF material NH2-Bi-BDC.

[0029] In one embodiment, the configuration of the glass fiber paper channel in step (1) is a glasses shape.

[0030] In one embodiment, the configuration of the glasses-shaped glass fiber paper channel in step (1) is as follows: the sample loading area and the fluorescent color development area are two circles with a diameter of 10 to 15 mm, connected in the middle by a narrow channel with a length of 3 to 5 mm and a width of 2 to 4 mm, and the extensions at both ends are 3 to 5 mm long and 2 to 3 mm wide.

[0031] In one embodiment, the configuration of the glasses-shaped glass fiber paper channel in step (1) is as follows: the sample loading area and the fluorescence color development area are two circles with a diameter of 13 mm, connected by a narrow channel with a length of 5 mm and a width of 2 mm in the middle, and the extensions at both ends are 4.5 mm long and 3 mm wide.

[0032] In one embodiment, the process of cutting the glasses-shaped paper channel in step (1) includes: using a laser engraving machine to engrave a paper channel model on an acrylic plate; and then placing the paper channel model on a glass fiber film to engrave the glasses-shaped paper channel.

[0033] In one embodiment, the preparation of the fluorescent MOF material NH2-Bi-BDC comprises the following steps:

[0034] 1) dissolving a soluble bismuth salt and 2-aminoterephthalic acid in N,N-dimethylformamide to prepare a mixed solution, which is then transferred to a polytetrafluoroethylene-lined steel autoclave for a hydrothermal reaction;

[0035] 2) The solution after the hydrothermal reaction in step (1) was naturally cooled to room temperature, centrifuged, washed, and dried to obtain the yellow solid fluorescent MOF material NH2-Bi-BDC.

[0036] In one embodiment, the soluble bismuth salt in step 1) includes one or more of bismuth nitrate, bismuth chloride, and bismuth subcarbonate.

[0037] In one embodiment, the molar ratio of the soluble bismuth salt to 2-aminoterephthalic acid in step 1) is 2:(1-4).

[0038] In one embodiment, the temperature of the hydrothermal reaction in step 1) is 100-150° C., and the time is 24-48 hours.

[0039] Another object of the present invention is to provide an application of the above-described method in the detection of glyphosate in water, soil, and food.

[0040] Advantages and effects of the present invention:

[0041] The present invention's NH2-Bi-BDC-based paper-based field-amplified enrichment and fluorescence detection method exhibits excellent glyphosate enrichment and fluorescence response capabilities. The electric field strength at different locations in the paper channel is negatively correlated with the channel width, effectively controlling the electric field strength through the eyeglass-shaped paper channel, which consists of two circular regions and a narrow central channel. However, glyphosate and the amino-functionalized MOF material are both negatively charged under the pH conditions of the background electrolyte and migrate in the same direction under the action of the electric field. Furthermore, due to the negative charge on the paper substrate surface, electroosmotic flow from the anode to the cathode is generated during electrophoresis. The present invention modulates the intensity of the electroosmotic flow by adding PVP to the cathode electrolyte, thereby achieving enrichment and interaction of glyphosate and the amino-functionalized MOF material within the paper channel. Furthermore, the selected fluorescent MOF material, NH2-Bi-BDC, possesses excellent electronic transition potential due to the flexible coordination structure of its central bismuth metal, enabling a rapid fluorescence response through hydrogen bonding interactions with glyphosate.

[0042] The present invention is used to detect glyphosate: the detection limit (LOD) is 0.2 μmol / L, the quantification limit (LOQ) is 0.8 μmol / L, and the two linear ranges are 0.8-25 and 25-200 μmol / L, respectively. In actual sample detection, 2.01 and 4.71 μmol / L of glyphosate were detected in soil and lake water samples, respectively. In a spiked recovery experiment, the spiked recovery rate was 95.74%-109.65%. The method is simple and rapid to operate, does not require large-scale instruments and equipment, and has high accuracy and good reproducibility. Therefore, it has great application prospects in on-site glyphosate monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the process of paper-based field amplified enrichment and fluorescence detection of glyphosate according to Example 2 of the present invention: A is a flow chart for the synthesis of NH2-Bi-BDC; B is a flow chart for the enrichment and fluorescence detection of glyphosate;

[0044] Figure 2 This is an environmental scanning electron micrograph of NH2-Bi-BDC prepared in Example 1 of the present invention;

[0045] Figure 3 This is the X-ray diffraction pattern of NH2-Bi-BDC prepared in Example 1 of the present invention;

[0046] Figure 4 This is a Fourier transform infrared spectrum of NH2-Bi-BDC prepared in Example 1 of the present invention;

[0047] Figure 5 This is the full X-ray photoelectron spectrum of NH2-Bi-BDC prepared in Example 1 of the present invention;

[0048] Figure 6This is the Bi 4f narrow spectrum of the X-ray photoelectron spectrum of NH2-Bi-BDC prepared in Example 1 of the present invention;

[0049] Figure 7 This is a fluorescence spectrum of NH2-Bi-BDC prepared in Example 1 of the present invention;

[0050] Figure 8 This is a schematic diagram of the geometric dimensions of the glasses-shaped paper channel in Example 2 of the present invention;

[0051] Figure 9 The actual pictures and optical microscope images of different paper-based materials in Comparative Example 2: A is a real picture of a glass fiber membrane; B is a real picture of a cellulose acetate membrane; C is an optical microscope image of a glass fiber membrane; D is an optical microscope image of a cellulose acetate membrane;

[0052] Figure 10 Comparison of the effects of different paper channel configurations on enrichment detection: A is a long strip paper channel; B is a glasses-shaped paper channel;

[0053] Figure 11 This is a comparison diagram of the effects of different PVP concentrations on enrichment detection in the present invention;

[0054] Figure 12 This is a comparison diagram of the effects of different BGE concentrations on enrichment detection in the present invention;

[0055] Figure 13 This is a comparison diagram of the effects of different enrichment times on enrichment detection in the present invention;

[0056] Figure 14 This is a comparison diagram of the effects of different NH2-Bi-BDC concentrations on enrichment detection in the present invention;

[0057] Figure 15 This is a standard curve diagram of the paper-based field amplification enrichment and fluorescence detection method for glyphosate in Example 3 of the present invention;

[0058] Figure 16 This is a comparison of the grayscale values ​​of the circular test strip without field amplification enrichment and GFA-PAD fluorescence imaging in Comparative Example 1. DETAILED DESCRIPTION

[0059] The present invention will be further described below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0060] Example 1: Synthesis of fluorescent metal organic framework material NH2-Bi-BDC

[0061] Dissolve 0.8152 g of 2-aminoterephthalic acid and 1.4552 g of bismuth nitrate pentahydrate in 60 mL of N,N-dimethylformamide. Stir at room temperature for 30 minutes, then transfer to a Teflon-lined steel autoclave and react at 120°C for 48 hours. After the reaction, cool naturally to room temperature and centrifuge the mixture at 8000 rpm to obtain a precipitate. The precipitate is washed with N,N-dimethylformamide and then ethanol, and finally dried under vacuum at 60°C for 12 hours to obtain a yellow solid, NH2-Bi-BDC.

[0062] Performance characterization of NH2-Bi-BDC materials:

[0063] The morphology and structure of the synthesized NH2-Bi-BDC were characterized by SEM. Figure 2 shown; Bi 3+ The NH2-Bi-BDC synthesized by coordination with 2-aminoterephthalic acid has a network morphology and is doped with spherical morphology, which preliminarily indicates the successful synthesis of a bismuth-based metal-organic framework.

[0064] The crystal structure of NH2-Bi-BDC was further explored by XRD. Figure 3 As shown in the figure, the synthesized NH2-Bi-BDC has obvious characteristic peaks at 2θ=27.14, 37.92, 39.60 and 48.68°, which are similar to those of H2-Bi-BDC, indicating that the introduction of amino groups does not affect the crystal configuration of bismuth terephthalate materials, further proving the successful synthesis of the material.

[0065] The functional groups and other properties of the synthesized NH2-Bi-BDC were investigated by FT-IR, such as Figure 4 As shown; at 3426 and 3349 cm -1 The characteristic peaks at 1545 and 828 cm are the stretching vibration peaks of NH, while the peaks at 1545 and 828 cm are the stretching vibration peaks of NH. -1 The characteristic peak at 1368 cm is pointed to the deformation vibration of NH, indicating that the amino group from NH2-BDC has been successfully introduced into the MOF material. -1 The characteristic peak at 544cm is the deformation vibration peak of CH; at the same time, -1 The characteristic peak at is attributed to Bi-O, indicating the successful synthesis of bismuth-based MOF materials.

[0066] The elements of the synthesized NH2-Bi-BDC were characterized by XPS, such as Figure 5 As shown: the peaks at binding energies of 159.3, 284.8, 400.0 and 531.4 correspond to Bi 4f, C 1s, N 1s and O1s orbitals, respectively, proving the presence of Bi atoms and N atoms in the composite material.

[0067] The Bi 4f of NH2-Bi-BDC was further characterized by narrow scan, such as Figure 6 As shown: After peak fitting, two main spectral peaks were obtained, located at 159.3 and 164.6 eV, respectively, representing Bi 4f7 and Bi 4f5 orbitals, which are consistent with the binding energy of Bi(Ⅲ), proving that Bi in the MOF material exists in a trivalent form.

[0068] The spectral properties of the synthesized NH2-Bi-BDC were investigated by UV lamp and fluorescence spectrometer. Figure 7 As shown: Digital camera photos taken under 365nm UV light show a significant increase in fluorescence intensity upon addition of glyphosate. Fluorescence spectrometer results are consistent with the digital photos. Scanning NH2-Bi-BDC using a Hitachi F-7000 fluorescence spectrometer at 365nm excitation wavelength reveals a distinct characteristic emission peak at 441nm. Adding 200μmol / L glyphosate to NH2-Bi-BDC increases the fluorescence intensity of this characteristic emission peak by 360%, accompanied by a red shift. This demonstrates the potential of NH2-Bi-BDC for fluorescence response and detection of glyphosate.

[0069] Example 2: Application of NH2-Bi-BDC in Paper-Based Field Amplified Enrichment and Fluorescence Detection of Glyphosate

[0070] Paper-based field amplified enrichment and fluorescence detection device for glyphosate Figure 1 As shown. Two electrolyte pools made of 10mL centrifuge tube caps were fixed to the plastic plate with double-sided tape. The glass fiber membrane was cut into two circular areas with a diameter of 13mm (the fluorescent color development area near the anode side and the sample loading area near the cathode side), connected in the middle by a narrow channel with a length of 5mm and a width of 2mm, and glasses-shaped paper channels with a length of 4.5mm and a width of 3mm at both ends (as shown in Figure 2). Figure 8 As shown), the two ends of the paper channel are placed in the anode and cathode electrolyte cells respectively; at the same time, two platinum wires are inserted into the anode and cathode electrolyte cells respectively as electrodes, and connected to a 200V DC power supply to form a pathway.

[0071] The process of enrichment detection is:

[0072] 400 μL of MOPS buffer (50 mmol / L, pH 6.5) was added to each of the two electrolyte cells as the anode and cathode electrolytes, respectively. 1% PVP was added to the cathode electrolyte to control electroosmotic flow. 20 μL of the glyphosate test solution was loaded into the sample area, and 20 μL of 0.025 g / L NH2-Bi-BDC prepared in Example 1 was loaded into the color development area as a fluorescent colorant. A constant voltage of 200 V was applied across the paper channel, causing glyphosate and NH2-Bi-BDC to migrate and accumulate under the influence of the electric field and electroosmotic flow. When glyphosate and NH2-Bi-BDC interact, a reaction occurs, enhancing the fluorescence signal and forming bands. Images were captured using a mobile phone under ultraviolet light. The resulting images were analyzed using Image J software for grayscale analysis. The grayscale ratio of the enhanced fluorescence bands before and after glyphosate addition was used as the basis for quantitative glyphosate analysis and calculation of the glyphosate content in the sample.

[0073] Example 3 Glyphosate Enrichment / Detection-Standard Curve

[0074] (1) Enrichment and fluorescence detection of standard samples: Glyphosate standards of different concentrations (concentrations were 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200 μmol / L) were treated with the glyphosate enrichment and fluorescence detection method of Example 2: 50 mmol / L MOPS buffer at pH = 6.5 was used as the background electrolyte, 1.0% PVP was added, 0.025 g / L NH2-Bi-BDC synthesized in Example 1 was used as the fluorescent color developer, the voltage was 200 V, and the enrichment time was 100 s.

[0075] (2) Constructing a standard curve:

[0076] The enriched paper channel was photographed under 365nm UV light and grayscale analysis was performed using Image J. The ratio of the grayscale value obtained to the blank group (G / G0) was used as the analytical signal of glyphosate and linearly correlated with the concentration of the glyphosate standard to analyze the linearity of the paper-based field-amplified enrichment and fluorescence detection method for glyphosate based on NH2-Bi-BDC (e.g. Figure 15 The effectiveness of this method was verified by analyzing characteristic parameters such as the limit of detection (LOD), the limit of quantification (LOQ), and reproducibility. The linear range of this method was 0.8-25 and 25-200 μmol / L, and the determination coefficient (R 2 ) were 0.9992 and 0.9977, LOD was 0.2 μmol / L, LOQ was 0.8 μmol / L, and the relative standard deviation (RSD) was 1.0% in nine parallel experiments, indicating that the method has good reproducibility.

[0077] Example 4 Detection of glyphosate in water samples and soil

[0078] Soil samples were collected from a farmland in Wuxi City, Jiangsu Province. Lake water and tap water were collected from Jiangnan University. Drinking water was purchased from a local supermarket. For soil samples, 1 g of sample was added to 40 mL of deionized water, sonicated for 30 minutes, and centrifuged at 4500 rpm for 10 minutes to obtain a supernatant. The supernatant was then filtered through a 0.22 μm filter membrane for subsequent glyphosate detection. For water samples, direct sampling was performed and filtered through a 0.22 μm filter membrane for subsequent glyphosate detection. Detection was performed using the method established in Example 2, and the glyphosate content in the test sample was calculated according to the calibration curve in Example 3.

[0079] The test results are shown in Table 1: Glyphosate was detected in soil and lake water samples at concentrations of 2.01 and 4.71 μmol / L, respectively, but not in tap water or drinking water.

[0080] In the spike recovery experiment, four samples were spiked with glyphosate standard solutions at varying concentrations (5 and 10 μmol / L), and each sample was measured five times. The glyphosate recoveries for the four samples ranged from 95.74% to 109.65%, with precisions (RSDs) less than 6.0%. This validates the accuracy of the developed method. See Table 1 for details.

[0081] Table 1 Glyphosate concentration (μmol / L), recovery (%) and precision (%, n=5) in actual samples

[0082]

[0083] Example 5 Optimization of enrichment detection conditions

[0084] (1) Selection of paper channel configuration:

[0085] Different geometric configurations of paper channels affect the electric field strength at different locations in the channel, thereby affecting the pH. The pH values ​​at various locations in a conventional long paper channel and a glasses-shaped paper channel were compared after field amplification and enrichment.

[0086] like Figure 10 A. The paper channel near the anode showed acidic results after extensive pH test paper testing, while the cathode showed strong alkalinity. Because NH2-Bi-BDC fluorescence detection of glyphosate is affected by pH and responds poorly under strong acidic and alkaline conditions, long paper channels are not suitable for field-amplified enrichment and fluorescence visualization analysis of glyphosate.

[0087] like Figure 10B. After field-amplified concentration in a spectacle-shaped paper channel, the pH of the paper channel remains largely stable between 6.0 and 7.0, providing a suitable pH environment for the reaction between glyphosate and NH2-Bi-BDC. Therefore, a spectacle-shaped paper channel was selected and combined with NH2-Bi-BDC to develop a paper-based analytical device for field-amplified concentration and fluorescence detection of glyphosate.

[0088] (2) Selection of polyvinylpyrrolidone (PVP) concentration:

[0089] The surface of the glass fiber membrane used in the paper channel is negatively charged, which will generate a strong electroosmotic flow (EOF) toward the cathode during field amplification. Therefore, a certain concentration of PVP needs to be added to the electrolyte as a thickener to suppress EOF. Since in this device, the analyte glyphosate and the color developer NH2-Bi-BDC are both negatively charged and move from the cathode to the anode under the action of the electric field, which is opposite to the direction of electroosmotic flow. In order to make the two meet in the color development area, it is necessary to effectively control EOF. Therefore, different concentrations of PVP were added to the cathode electrolytic cell, and the effect of PVP on the enrichment effect was explored by comparing the grayscale values ​​at different positions after field amplification. The results are shown in Figure 2. Figure 11 .

[0090] As can be seen from the results in the figure, when no PVP was added, no obvious accumulation band was observed; when a certain amount of PVP was added to the electrolyte, a fluorescence enhancement band was observed, and as the PVP concentration increased, the fluorescence enhancement area moved toward the anode. When the PVP concentration was 3.0%, because the electric field effect was much greater than the EOF, NH2-Bi-BDC entered the anode electrolytic cell under the drive of the electric field, and the enrichment effect was poor. According to the grayscale analysis of the image obtained by a smartphone under ultraviolet light, when the PVP was 1.0%, the grayscale value of the corresponding fluorescence enhancement area reached 142, which was much higher than the other groups (0.5%, 125.2; 2%, 120.4). The field amplification enrichment effect was better, so the PVP concentration was preferably 1.0%.

[0091] (3) Selection of background electrolyte (BGE) concentration:

[0092] During the field-amplified electrokinetic deposition process, water electrolysis reaction is accompanied by the generation of H + and OH - , which in turn affects the pH of the electrolyte, so MOPS buffer was used to maintain the stability of pH conditions. When 1.0% PVP was added, the effect of different concentrations of MOPS buffer on glyphosate enrichment was studied. The results are as follows Figure 12 As shown;

[0093] The results in the figure show that the signal intensity of sample enrichment increases with increasing BGE concentration. However, when the BGE concentration exceeds 50 mmol / L, the signal intensity gradually decreases. This is because excessive BGE concentration increases the electric field driving force, which cannot compete with the electroosmotic flow, thus affecting the enrichment effect. Therefore, 50 mmol / L MOPS was selected as the electrolysis buffer.

[0094] (4) Selection of enrichment time:

[0095] Under the optimized PVP and BGE concentration conditions, the time-dependent changes in the intensity of the accumulation band were studied. Figure 13 No distinct bands were observed in the first 60 seconds. After 60 seconds, an enriched band gradually emerged with increasing fluorescence. The intensity of the enriched band stabilized after 100 seconds, so images after 100 seconds of field magnification and accumulation were selected for quantitative analysis.

[0096] (5) Selection of NH2-Bi-BDC concentration:

[0097] The concentration of fluorescent MOF material NH2-Bi-BDC affects the enrichment and color development effect. The grayscale value of the fluorescence enhancement area of ​​the paper channel after 100s field amplification enrichment with different concentrations of NH2-Bi-BDC was investigated. The results are as follows: Figure 14 As shown:

[0098] The results in the figure show that when the NH2-Bi-BDC concentration was 0.01 g / L, no enrichment bands appeared in the glyphosate-added group. When the concentration exceeded 0.025 g / L, a region of fluorescence enhancement appeared in the color development area. However, at higher concentrations, the fluorescence enhancement area expanded, and a clear crescent-shaped band could not be formed. Compared with the blank group, the NH2-Bi-BDC concentration of 0.025 g / L achieved the best light enhancement effect before and after glyphosate addition. However, at higher concentrations, the color developer concentration was too high, affecting the sensitivity of the color development reaction. Therefore, an NH2-Bi-BDC concentration of 0.025 g / L was selected.

[0099] Comparative Example 1

[0100] The difference from Example 2 is that field amplification enrichment is not used. 20 μL of 0.025 g / L NH2-Bi-BDC prepared in Example 1 is directly dropped onto a circular test paper with a diameter of 13 mm. After drying, 20 μL of glyphosate standard solutions with concentrations of 10 μmol / L, 100 μmol / L, 1 mmol / L, and 10 mmol / L are added respectively. The average grayscale value after imaging is compared with the enrichment and imaging grayscale results of 20 μL of 0.8 μmol / L glyphosate standard solution added after GFA-PAD.

[0101] The results are as follows Figure 16 As shown, when the 0.8 μmol / L glyphosate standard sample was enriched by GFA-PAD for 100 seconds, the grayscale value of the band reached the highest level and exceeded the reference level of 10 mmol / L in Comparative Example 1 (dashed line in the figure). This result indicates that the signal intensity was enhanced by GFA-PAD by approximately 12,500 times.

[0102] In addition, the imaging photos of Comparative Example 1 under 365nm UV light ( Figure 16 As can be seen in the illustration (see Figure 2), because the capillary force on the paper substrate is nondirectional, the probes on the test paper are dislodged when the sample solution is added, resulting in uneven fluorescence enhancement. However, the GFA-PAD allows for directional movement of the probe and target under the control of an electric field and electroosmotic flow, enabling stable fluorescence visualization of glyphosate.

[0103] Comparative Example 2

[0104] In the preliminary experiment, the difference from Example 2 is that the glass fiber membrane of the paper-based material is replaced with the cellulose acetate membrane. Figure 9 Shown (A, glass fiber membrane; B, cellulose acetate membrane) and optical microscope imaging (C, glass fiber membrane; D, cellulose acetate membrane).

[0105] Since cellulose acetate membrane has poor water retention, it evaporates during the electrophoresis process, resulting in the inability to display the fluorescence enhancement effect. Therefore, cellulose acetate membrane cannot be used as a paper-based material for field amplification.

[0106] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for glyphosate based on paper-based field amplification enrichment and fluorescence detection, characterized in that: The method comprises the following steps: (1) Formation of fluorescent enrichment bands A glass fiber membrane is used as a paper channel, and together with a DC high-voltage power supply, an electrolyte cell, and a platinum wire electrode, a paper-based field amplification device is formed. The platinum wire electrode is immersed in the cathode and anode electrolyte cells, respectively, and polyvinyl pyrrolidone is added to the cathode electrolyte cell. The paper channel is sequentially divided into a sample loading area, a sample enrichment area, and a fluorescence color development area. The fluorescence color development area is located near the anode side, and the sample loading area is located near the cathode side. A sample containing glyphosate is dripped into the sample loading area, and a fluorescence color developer is dripped into the fluorescence color development area. A constant voltage is applied to cause glyphosate to move through the sample enrichment area for enrichment. When the enriched glyphosate meets NH2-Bi-BDC, a reaction occurs, resulting in an enriched band with enhanced fluorescence in the fluorescence color development area. The fluorescence color developer is a fluorescent MOF material NH2-Bi-BDC. The concentration of NH2-Bi-BDC is 0.025-0.1 g / L. (2) Determination of glyphosate content According to step (1), a series of glyphosate standard samples with known concentrations and the fluorescence enrichment color bands of the glyphosate sample to be tested are obtained respectively, and the image information is obtained by photographing and imaging under ultraviolet light, and grayscale analysis is performed. The grayscale ratio of the fluorescence enhanced band before and after the addition of glyphosate is used as the basis for the quantitative analysis of glyphosate, and a linear relationship between the fluorescence enhanced band and the glyphosate concentration is constructed to obtain a glyphosate standard curve; at the same time, based on the standard curve, the glyphosate content in the glyphosate sample to be tested is calculated.

2. The method according to claim 1, characterized in that The mass concentration of the polyvinyl pyrrolidone in step (1) relative to the cathode electrolyte is 0.5-2.0%.

3. The method according to claim 1, characterized in that The preparation of the fluorescent MOF material NH2-Bi-BDC includes the following steps: 1) Dissolving a soluble bismuth salt and 2-aminoterephthalic acid in N,N-dimethylformamide to prepare a mixed solution, which is then transferred to a polytetrafluoroethylene-lined steel autoclave for a hydrothermal reaction; 2) The solution after the hydrothermal reaction in step (1) is naturally cooled to room temperature, centrifuged, washed, and dried to obtain the yellow solid fluorescent MOF material NH2-Bi-BDC.

4. The method according to claim 1, wherein The electrolyte in the electrolyte cell of step (1) is a 3-(N-morpholino)propanesulfonic acid buffer solution with a pH of 6.5 and a concentration of 25-75 mmol / L.

5. The method according to claim 1, wherein The enrichment time in step (1) is 60 to 160 s.

6. The method according to claim 1, wherein The paper channel in step (1) is a glasses-type paper channel configuration. The specific parameters of the glasses-type paper channel configuration are: the sample loading area and the fluorescence color development area are two circles with a diameter of 10-15 mm, connected in the middle by a narrow channel with a length of 3-5 mm and a width of 2-4 mm, and the extensions at both ends are 3-5 mm long and 2-3 mm wide.

7. The method according to claim 1, wherein The wavelength of the ultraviolet lamp irradiation in step (2) is 365 nm.

8. The method according to any one of claims 1 to 7, characterized in that The mass concentration of the polyvinyl pyrrolidone in step (1) relative to the cathode electrolyte is 1.0%.

9. Use of the method according to any one of claims 1 to 8 in the detection of glyphosate in water, soil and food.

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