Method for detecting glyphosate based on ratio-type fluorescent test paper

Abstract

The application discloses a method for detecting glyphosate based on a ratio type fluorescent test paper sheet, the test paper sheet comprising a paper base material, a fluorescent annular sticker and a probe material, wherein the fluorescent annular sticker is arranged on the paper base material as an annular reference area, a central circular area formed by the annular reference area is a central sample area, and the central sample area is previously added with NH2-Bi-MOF / Cu 2+ As the probe material, after drying, NH2-Bi-MOF / Cu 2+ The ratio type fluorescent test paper sheet of the reaction system. The sample to be detected is added to the central sample area of the ratio type fluorescent test paper sheet, and after reaction, the glyphosate concentration is semi-quantitatively analyzed by comparison with a standard color card under ultraviolet lamp irradiation, or, picture information is obtained by imaging under ultraviolet lamp irradiation, and the glyphosate concentration is calculated according to a ratio type quantitative detection model by comparing the gray values of the central sample area and the annular reference area. The method has the advantages of high speed, convenient operation and practical application prospect, and can be used for detecting glyphosate in water and food samples.

Description

Technical Field

[0001] This invention relates to a method for detecting glyphosate based on ratiometric fluorescent test strips, belonging to the field of analytical detection technology. Background Technology

[0002] Glyphosate, an organophosphorus herbicide developed by Monsanto in the 1980s, is widely used in agriculture for weed control due to its excellent systemic conductivity within plants. Since the early 1990s, glyphosate usage has increased by over 500%, with the annual application area equivalent to 30%–40% of total arable land. Although some glyphosate in the environment degrades through biological or photochemical pathways, some glyphosate and its metabolites remain, accumulating in the food chain. Despite ongoing debate regarding the toxicity of glyphosate, growing public concern about environmental and food safety has created a demand for glyphosate residue analysis. Furthermore, given the short shelf life of agricultural products, the analytical procedures for on-site detection of glyphosate in food must be time-efficient, and the analytical devices must be readily available and portable, while simultaneously ensuring reliable analytical accuracy in complex environments.

[0003] Ratio sensors are analytical tools that reduce errors from the environment and equipment by using built-in self-calibrating references. In recent years, they have been increasingly developed for the rapid detection of targets in complex and variable environments. Based on different output signal modes, ratio sensors can be divided into four main categories: temperature sensors, electrochemical sensors, colorimetric sensors, and fluorescence sensors. Among them, fluorescence-based analytical methods have advantages such as short turnaround time and simple preprocessing, and they exhibit higher sensitivity in visual inspection compared to colorimetric sensors. These sensors demonstrate stable analytical performance in complex environments through ratio detection strategies, but they still require specialized instruments for signal output. Developing portable analytical devices based on ratiometric fluorescence sensing strategies is beneficial for achieving more accurate and convenient on-site detection of analytes.

[0004] In recent years, paper-based analytical devices (PADs) have been widely used for on-site environmental and food safety testing due to their advantages such as visualization capabilities (not requiring specialized fluorescence spectrometers) and ease of acquisition, manufacturing, and portability. However, PADs typically rely on imaging to acquire signals, and their dependence on angle and light can easily introduce errors, thus reducing the accuracy of their analysis and introducing uncertainties in practical applications. Therefore, applying ratiometric sensing strategies to PADs has great potential for on-site detection and analysis. Traditional ratiometric fluorescence sensors achieve ratiometric sensing strategies by synthesizing dual-emission probe materials. This requires that the peak positions of the dual-emission probes do not interfere with each other, increasing the design difficulty during material synthesis. Furthermore, since the naked eye cannot accurately distinguish the fluorescence signals of the target and the reference, such PADs based on dual-emission probe materials cannot be used for rapid visual inspection. How to achieve regional signal output for the target and the reference is particularly important for constructing PADs that combine rapid visual inspection and high-precision ratio analysis capabilities. Summary of the Invention

[0005] To address the aforementioned issues, this invention uses a glass fiber membrane as the paper-based material, attaches a fluorescent ring-shaped sticker to it, and pre-drops NH2-Bi-MOF / Cu into the central sample area. 2+ A ratiometric fluorescent test strip was constructed as a probe material, providing a method for the rapid semi-quantitative detection of glyphosate via colorimetric cards or the comparative quantitative analysis of glyphosate using grayscale values ​​through imaging. This method is fast, convenient, and has promising practical applications for the detection of glyphosate in water and food samples.

[0006] The first objective of this invention is to provide a method for rapid detection of glyphosate using a fluorescence sensor, comprising the following steps:

[0007] (1) The fluorescent MOF material NH2-Bi-MOF was dispersed in water and the pH was adjusted to weakly acidic or neutral. Cu was then added. 2+ NH2-Bi-MOF / Cu 2+ Sensing system;

[0008] (2) A certain concentration of glyphosate standard sample is added to the sensing system, and the ratio of fluorescence intensity before and after the addition of standard sample is used as the signal output. The ratio is linearly correlated with the glyphosate concentration to obtain a quantitative detection model.

[0009] (3) The sample to be tested is added to the sensing system, and the ratio of fluorescence intensity before and after the sample to be tested is used as the signal output. By referring to the quantitative detection model, the corresponding glyphosate concentration is obtained.

[0010] In one embodiment of the present invention, the fluorescent MOF material NH2-Bi-MOF is synthesized by a hydrothermal method.

[0011] In one embodiment of the present invention, the concentration of NH2-Bi-MOF in the sensing system is 0.025-0.5 g / L. -1 Preferably 0.25g L -1 .

[0012] In one embodiment of the present invention, Cu in the sensing system 2+ Concentration of 80-100 μmol L -1 Preferably 90 μmol L -1 This invention selects Cu 2+ As a quencher, Cu combines with NH2-Bi-MOF to form a sensing system because... 2+ It can undergo a Lewis acid-base reaction with the amino groups on the surface of NH2-Bi-MOF, thereby quenching the latter's fluorescence signal. The amount of quencher added directly affects the sensitivity and linear range of this method.

[0013] In one embodiment of the present invention, the pH of the sensing system is 5.0-7.0, preferably 6.0.

[0014] In one embodiment of the present invention, the fluorescence intensity of the sensing system 0-5 min after the sample is added is used as the signal output, preferably the fluorescence intensity of the system 1 min after the sample is added is used as the signal output.

[0015] In one embodiment of the present invention, in the method for rapid detection of glyphosate using the fluorescent sensor, the detection limit for glyphosate is 0.05 μmol / L. -1 .

[0016] The second objective of this invention is to provide a sensing system for the rapid detection of glyphosate using a fluorescence sensor, wherein the sensing system is NH2-Bi-MOF / Cu. 2+ The sensing system consists of fluorescent MOF material NH2-Bi-MOF dispersed in water with the pH adjusted to weakly acidic or neutral, followed by the addition of Cu. 2+ get.

[0017] The third objective of this invention is to provide a ratiometric fluorescent test strip, comprising a paper base material, a fluorescent ring sticker, and a probe material. The fluorescent ring sticker is disposed on the paper base material as a ring-shaped reference region, and the resulting central circular region is the central sample region, i.e., the sample area. The central sample region is pre-added with NH2-Bi-MOF / Cu. 2+ As a probe material, after drying, it forms an NH2-Bi-MOF / Cu structure. 2+Ratio-type fluorescent test strips for the reaction system.

[0018] In one embodiment of the present invention, the preparation of the test strip includes the following process:

[0019] (1) Cut square test paper pieces from paper-based materials such as glass fiber membrane, punch holes in adhesive fluorescent roll paper to obtain fluorescent ring stickers, and assemble the fluorescent ring stickers on top of the glass fiber membrane test paper.

[0020] (2) The fluorescent MOF material NH2-Bi-MOF was synthesized by hydrothermal reaction, and Cu was added in advance. 2+ NH2-Bi-MOF / Cu 2+ The reaction system was added dropwise to the sample area of ​​the test strip, and after drying, a ratiometric fluorescent test strip was obtained.

[0021] In one embodiment of the present invention, the paper-based material includes any one of glass fiber membrane, Whatman No.1 paper filter, and cellulose acetate membrane, preferably glass fiber membrane.

[0022] In one embodiment of the present invention, the fluorescent ring sticker is adhered to the paper-based material.

[0023] In one embodiment of the present invention, the paper base material is square, and its side length is greater than or equal to the outer diameter of the fluorescent ring sticker. Preferably, the side length of the paper base material is equal to the outer diameter of the fluorescent ring sticker, which makes the size ratio more aesthetically pleasing.

[0024] In one embodiment of the present invention, the ratio of the outer diameter to the inner diameter of the fluorescent ring sticker is (4-10):3, preferably 5:3.

[0025] In one embodiment of the present invention, the inner diameter of the annular sticker in step (1) is 6 mm and the outer diameter is 10 mm. The inner diameter of 6 mm is matched with the sample volume of 2 μL, and the size of the inner diameter is adjusted according to the sample volume.

[0026] In one embodiment of the present invention, the side length of the square test strip in step (1) is 10 mm. In other embodiments, the outer diameter can theoretically be greater than 10 mm.

[0027] In one embodiment of the present invention, the concentration of NH2-Bi-MOF in the reaction system in step (2) is 0.025-1.0 g / L. -1 Preferably, it is 0.025-0.5g / L. -1 More preferably 0.25g L -1 .

[0028] In one embodiment of the present invention, Cu in the reaction system in step (2)2+ Concentration of 80-100 μmol L -1 Preferably 90 μmol L -1 .

[0029] In one embodiment of the present invention, the volume of the reaction system added to the sample area in step (2) is 1-5 μL, preferably 2 μL.

[0030] In one embodiment of the present invention, the detection limit of the ratiometric fluorescent test strip for glyphosate is 0.82 μmol / L. -1 .

[0031] The fourth objective of this invention is to provide a method for detecting glyphosate using a ratiometric fluorescent test strip, comprising the following steps:

[0032] (1) The sample to be tested is dropped onto the sample area of ​​the ratio-type fluorescent test strip;

[0033] (2) After the reaction, the glyphosate concentration was semi-quantitatively analyzed by comparing it with the standard colorimetric card under ultraviolet light irradiation, and / or, the image information was obtained by taking pictures under ultraviolet light irradiation and grayscale analysis was performed. The grayscale values ​​of the central sample area and the annular reference area were compared, and the corresponding glyphosate concentration was calculated according to the ratio-type quantitative detection model. The glyphosate concentration was then quantitatively analyzed.

[0034] The standard colorimetric card is a standard concentration reference model constructed by linearly correlating the results of standard samples with known concentration gradients with glyphosate concentrations.

[0035] The ratiometric quantitative detection model is a standard curve constructed by linearly correlating the results of standard samples with known concentration gradients with glyphosate concentration.

[0036] In one embodiment of the present invention, the quantitative analysis of glyphosate concentration in step (2) specifically involves: obtaining image information by taking a picture with a mobile phone under ultraviolet light; performing grayscale analysis using ImageJ software, comparing the grayscale values ​​of the sample area with those of the annular reference area, and linearly correlating them with the glyphosate concentration to obtain a ratio-type quantitative detection model.

[0037] In one embodiment of the present invention, the volume of glyphosate-containing sample added to the sample area in step (1) is 1-5 μL, preferably 2 μL.

[0038] In one embodiment of the present invention, the standard colorimetric card in step (2) is divided into five concentration reference values, wherein Nd is undetectable (below 1 μmol / L). -1 ), Tr represents trace amounts (1-5 μmol L). -1 Lo is a small amount (5-10 μmol L). -1Md is moderate (10-100 μmol L). -1 Hi is a large amount (more than 100 μmol L). -1 ).

[0039] In one embodiment of the present invention, the wavelength of the ultraviolet lamp irradiation is 365nm.

[0040] In one embodiment of the present invention, in step (2), the test strip after 0-5 minutes of adding the sample to be tested is photographed and analyzed. Preferably, the test strip after 1 minute of adding the sample to be tested is photographed and analyzed.

[0041] In one embodiment of the present invention, in step (2), the image acquisition is performed using an image acquisition device such as a mobile phone or a camera.

[0042] A fifth objective of the present invention is to provide a detection device comprising a sensing system as described above, or a ratiometric fluorescent test strip as described above.

[0043] In one embodiment of the present invention, the detection device is any usable device form, including a reagent kit.

[0044] The sixth objective of this invention is to provide the application of the sensing system and detection method described above, or the ratiometric fluorescent test strip and detection method described above, or the detection device described above, in the detection of glyphosate in water, soil, and food.

[0045] Advantages and effects of the present invention:

[0046] (1) The present invention is based on NH2-Bi-MOF / Cu 2+ The ratiometric fluorescent test strip method for detecting glyphosate is rapid and convenient, and has good resistance to environmental interference such as imaging light and angle. Fluorescent test strips have advantages such as portability and economy, and have great application prospects in on-site detection of pesticide residues. However, because fluorescent imaging is easily affected by environmental factors such as light and angle, the accuracy of the analysis is affected.

[0047] (2) This invention designs a ratio-type fluorescent test strip by attaching fluorescent stickers to the test strip. By comparing the gray values ​​of the sample area and the reference area, a more accurate quantitative analysis of glyphosate is achieved. At the same time, the regional design of the sample signal and the reference signal can achieve rapid semi-quantitative visual detection with the comparison of a standard colorimetric card.

[0048] (3) Furthermore, in this invention, NH2-Bi-MOF and glyphosate exhibit highly efficient fluorescence response capabilities, and the quencher Cu... 2+The addition of [a specific ingredient] increases the linear range of detection. Using the ratiometric fluorescent test strip of this invention to detect glyphosate: the relationship between G / G0 and glyphosate concentration conforms to a Logistic function, R[…]. 2 The value was 0.9984. In the actual sample detection, 4.40 μmol / L glyphosate was detected in soybean samples. In the spiked recovery experiments of soybean, wheat and drinking water samples, the spiked recovery rate was 97.6% to 109.9%, and the results were consistent with the results of solution sensor and liquid chromatography-mass spectrometry (LC-MS) method.

[0049] (4) This method is low in cost, easy to operate, and quick to analyze. It does not require large instruments and equipment, and has high accuracy and good stability. It has great application prospects in the field monitoring of glyphosate. Attached Figure Description

[0050] Figure 1 Flowchart for ratiometric fluorescent test strip detection of glyphosate: A represents the synthesis of NH2-Bi-MOF and the process based on NH2-Bi-MOF / Cu. 2+ Schematic diagram of fluorescence sensor detection; B represents the detection based on NH2-Bi-MOF / Cu. 2+ A schematic diagram of the construction and detection of ratio-type fluorescent test strips; C is a schematic diagram of the structure of ratio-type fluorescent test strips.

[0051] Figure 2 Fluorescence spectrum of NH2-Bi-MOF;

[0052] Figure 3 Fourier transform infrared spectrum of NH2-Bi-MOF;

[0053] Figure 4 X-ray photoelectron spectroscopy of NH2-Bi-MOF with a narrow Cu 2p spectrum;

[0054] Figure 5 Comparison of the effects of different concentrations on the fluorescence intensity of NH2-Bi-MOF;

[0055] Figure 6 NH2-Bi-MOF / Cu 2+ Standard curve for glyphosate detection using a fluorescence sensor;

[0056] Figure 7 NH2-Bi-MOF / Cu 2+ Selectivity for glyphosate;

[0057] Figure 8 NH2-Bi-MOF / Cu 2+ Its anti-interference performance;

[0058] Figure 9Fluorescence spectrum of the sticker;

[0059] Figure 10 Comparison of stability between conventional and ratio-type test strips: A is conventional fluorescent test strip; B is ratio-type fluorescent test strip.

[0060] Figure 11 Comparison of the effects of different paper-based materials on fluorescence sensing performance;

[0061] Figure 12 Comparison of the effects of different NH2-Bi-MOF concentrations on fluorescence sensing performance: A is an actual image under a 365nm UV lamp; B is a graph showing the relationship between G / G0 and NH2-Bi-MOF concentration.

[0062] Figure 13 Comparison of the effects of different response times on fluorescence sensing performance: A is a picture of the actual object under a 365nm UV lamp; B is a graph showing the relationship between G / G0 and response time.

[0063] Figure 14 Schematic diagram of a standard colorimetric card;

[0064] Figure 15 Standard curve for the detection of glyphosate using ratiometric fluorescent test strips. Detailed Implementation

[0065] The present invention will be further described below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0066] Example 1: NH2-Bi-MOF / Cu 2+ Fluorescent sensor detects glyphosate

[0067] NH2-Bi-MOF / Cu 2+ The process of detecting glyphosate with a fluorescence sensor is as follows: Figure 1 As shown in Figure A. First, a uniformly dispersed NH₂-Bi-MOF solution (0.25 g / L) was prepared using ultrapure water (pH 6.0). -1 (The preparation method of NH2-Bi-MOF is not limited in this invention), and the mixture is ultrasonically treated for 20 minutes. Then, Cu is added. 2+ (90 μmol L) -1 The fluorescence-quenched sensor solution was obtained. Then, glyphosate standard solution (0–200 μmol / L) was added. -1 After 1 minute, the fluorescence spectrum was recorded, with the excitation wavelength set to 365 nm. A quantitative model was constructed by linearly correlating the concentration of glyphosate with the ratio of the corresponding response fluorescence intensity to that of the blank group (I / I0).

[0068] NH2-Bi-MOF / Cu 2+ Fluorescence properties and sensing mechanism:

[0069] To investigate the mechanism of the fluorescence response of NH2-Bi-MOF to glyphosate, the fluorescence response of NH2-Bi-MOF in the presence of Cu was first examined. 2 + Fluorescence spectra under glyphosate conditions. For example... Figure 2 As shown, NH2-Bi-MOF exhibits a characteristic fluorescence peak at 441 nm. The addition of Cu... 2+ Afterward, the fluorescence signal was quenched by approximately 64%. Subsequently, the addition of glyphosate resulted in a 23 nm redshift in the emission spectrum of NH2-Bi-MOF, and a 576% enhancement in the fluorescence signal. In digital photographs taken under ultraviolet light, the previously almost non-fluorescent NH2-Bi-MOF / Cu... 2+ system( Figure 2 Illustration b) begins to emit a strong blue fluorescence in the presence of glyphosate. Figure 2 Illustration c) demonstrates the feasibility of its visual detection.

[0070] Further investigation of the mechanism using FT-IR, such as Figure 3 As shown. The FT-IR spectra of NH2-Bi-MOF are at 1361 and 830 cm⁻¹. -1 Characteristic peaks appear nearby, at 3438 and 3346 cm⁻¹. -1 There are slight splitting peaks nearby, pointing to the deformation and stretching vibrations of NH, respectively, while at 503 cm⁻¹... -1 The peaks at these points point to the metal-oxygen bond Bi-O, indicating the successful synthesis of NH2-Bi-MOF. The addition of Cu... 2+ After adding glyphosate, the 3400cm² representing the amino group... -1 The nearby splitting peaks gradually weaken until they merge with the peaks generated by OH stretching, indicating that NH2-Bi-MOF reacts with Cu. 2+ The interaction with glyphosate is primarily based on amino groups. To further investigate Cu... 2+ The binding properties with NH2-Bi-MOF and glyphosate, and the effect of NH2-Bi-MOF / Cu 2+ The Cu 2p XPS narrow spectrum before and after the addition of glyphosate was studied, as follows: Figure 4 As shown. NH2-Bi-MOF / Cu 2+ Peaks were observed at 932.5 and 952.2 eV, respectively, consistent with Cu 2p. 3 / 2 and Cu 2p 1 / 2 The binding energy of Cu. The addition of glyphosate increased the corresponding binding energy by 0.8 eV, indicating that Cu... 2+ The binding with glyphosate is more stable, further proving the role of Cu. 2+ The mediated "on-off-on" response strategy is reliable.

[0071] Cu2+ Concentration selection:

[0072] This invention selects Cu 2+ As a quencher, Cu 2+ The quencher can undergo a Lewis acid-base reaction with the amino groups on the surface of NH2-Bi-MOF, thereby quenching the latter's fluorescence signal. The amount of quencher added directly affects the sensitivity and linear range of this method. This example investigates the effect of Cu... 2+ The trend of changes in the amount of NH2-Bi-MOF added and the fluorescence intensity before and after addition ( Figure 5 ).

[0073] As can be seen from the figure, with Cu 2+ With increasing addition, the fluorescence intensity of NH2-Bi-MOF gradually decreased, and the fluorescence intensity ratio I / I0 before and after addition also showed a decreasing trend. When Cu... 2+ The amount added was 90 μmol L. -1 The fluorescence quenching effect is optimal at this time. Therefore, in subsequent embodiments, Cu is preferentially selected. 2+ The amount added was 90 μmol L. -1 .

[0074] NH2-Bi-MOF / Cu 2+ Quantitative model for glyphosate detection:

[0075] To evaluate the established NH2-Bi-MOF / Cu 2+ A fluorescent sensor method for detecting glyphosate, using NH2-Bi-MOF (2.5 × 10⁻⁶) at pH 6. -2 g L -1 ) and Cu 2+ (90 μmol L) -1 Aqueous solution. Subsequently, in NH2-Bi-MOF / Cu 2+ Different concentrations of glyphosate standard solutions (0–200 μmol / L) were added to the system. -1 ), scan fluorescence spectrum.

[0076] The results are as follows Figure 6 As shown, after the addition of glyphosate, the fluorescence ratio of I / I0 was related to the glyphosate concentration in the range of 0.1–30 μmol / L. -1 (R 2 =0.9922) and 30~200μmol L -1 (R 2 It exhibits good linearity in the two ranges (=0.9917), and also shows good linearity in the concentration range of 0.1–30 μmol L. -1At this time, it exhibits higher precision (I / I0 = 0.0971c + 1.0795). In this method, the LOD value of glyphosate is 0.05 μmol L⁻¹. -1 .

[0077] NH2-Bi-MOF / Cu 2+ Selectivity and anti-interference performance:

[0078] With metal ions (Pb) 2+ Cd 2+ Na + K + Ba 2+ 900 μmol L -1 ) and organophosphorus pesticides (parathion, parathion methyl, promethazine, phosphazene, phosphate, 30 μmol L -1 Further investigation was conducted on NH2-Bi-MOF / Cu as a disruptor. 2+ glyphosate (30 μmol L) -1 The selectivity of ) results as follows Figure 7 As shown in the photos taken under UV light, only the group with added glyphosate exhibited strong blue fluorescence. Correspondingly, only the group with added glyphosate showed a 329% increase in the fluorescence intensity ratio (I / I0) before and after adding glyphosate, while the signal changes in other groups were minimal.

[0079] Further investigation is needed into the system's anti-interference performance, such as... Figure 8 As shown. Pre-treated in NH2-Bi-MOF / Cu 2+ The aforementioned interfering agents were loaded, followed by the addition of glyphosate, and the corresponding fluorescence spectra were recorded. All experimental groups showed the same fluorescence characteristics as the blank group (NH2-Bi-MOF / Cu). 2+ The enhanced fluorescence signal, similar to that of the system, indicates that the system has good anti-interference performance.

[0080] Example 2: NH2-Bi-MOF / Cu 2+ Application in the detection of glyphosate using ratiometric fluorescent test strips

[0081] The process of detecting glyphosate using ratiometric fluorescent test strips is as follows: Figure 1 As shown in B. First, cut the glass fiber membrane to obtain square test paper strips with a side length of 10 mm. Select adhesive roll paper with a fluorescent signal; its fluorescence spectrum is as follows. Figure 9 As shown. A fluorescent hydrophobic ring-shaped sticker with an inner diameter of 6 mm and an outer diameter of 10 mm was obtained using a punch. This sticker was then adhered to the surface of a glass fiber film test strip as a reference. The test strip structure is as follows. Figure 1 As shown in C, the fluorescent ring sticker is the ring reference area, and the central circular area formed is the central sample area.

[0082] Then, 2 μL of NH2-Bi-MOF / Cu 2+ (NH2-Bi-MOF: 0.25g L) -1 Cu 2+ 90 μmol L -1 A drop was added to the central sample area, and after drying, a ratiometric fluorescent test paper was obtained. Then, 2 μL of glyphosate (0–200 μmol / L) was added. -1 The sample area exhibits enhanced blue fluorescence, which can be used for semi-quantitative detection by visual visualization. A digital photograph is taken under 365nm UV light after 1 minute, and grayscale analysis is performed using ImageJ. The grayscale ratio of the sample area and the reference area is calculated as the signal output. A quantitative model is constructed by linearly correlating the glyphosate concentration with the grayscale ratio (G / G0) of the sample and reference areas for quantitative detection.

[0083] Comparative Example 1

[0084] The difference from Example 2 is that the ratiometric fluorescent test strip is replaced with a traditional fluorescent test strip (i.e., only the gray value of the sample area is read as the signal output). Parallel experiments were conducted to verify whether the ratiometric fluorescent test strip in this invention can reduce the analytical errors caused by imaging angle and lighting conditions.

[0085] like Figure 10 As shown in Figure A, traditional fluorescent test strips only output the grayscale value of the sample area as a signal. This results in signals for each concentration spanning a large range, easily causing confusion with results from adjacent concentrations. Therefore, the accuracy of traditional fluorescent test strips may be questionable in practical applications. However, when using the ratiometric fluorescent test strip of this invention, as... Figure 10 As shown in Figure B, the results of the six concentration gradients are more concentrated than those of traditional fluorescent test strips, indicating its promising application prospects in the on-site detection of glyphosate residues.

[0086] Example 3: Optimization of Detection Conditions

[0087] (1) Selection of paper base material:

[0088] The material of the paper base can affect the fluorescence imaging effect of the test strip, thus affecting the test results. In order to select a suitable paper base material, glass fiber membrane, Whatman No.1 paper filter, and cellulose acetate membrane were compared.

[0089] The effects of glyphosate (100 μmol L) on glyphosate (100 μmol L) were investigated. -1 Fluorescence phenomena on test papers with different paper base materials before and after addition, such as Figure 11As shown in the figure, all three groups of paper-based materials exhibit fluorescent spots with strong signals, indicating that all three paper-based materials are applicable. Furthermore, it can be seen that the glass fiber membrane (group a) exhibits uniform fluorescent spots with strong signals, while in groups b and c, the radial flow of liquid on the paper surface carries solute particles to the edge of the droplets, resulting in a "coffee ring" phenomenon, which affects the uniformity of the fluorescent spots. Therefore, in subsequent embodiments, glass fiber membranes are preferentially selected as the paper-based material for constructing ratiometric fluorescent test strips.

[0090] (2) Selection of NH2-Bi-MOF concentration:

[0091] The concentration of the fluorescent probe affects the efficiency of its interaction with the target analyte, thus affecting the sensitivity of the test strip. Therefore, the effect of NH2-Bi-MOF concentration on the addition of glyphosate (5 μmol L) was investigated. -1 The effect of Cu on the fluorescence properties of the test strips before and after the test. 2+ The concentration was set to 90 μmol L. -1 .

[0092] Based on visual observation ( Figure 12 A) Differences in fluorescence intensity were observed before and after the addition of glyphosate, especially with a difference of 0.25 g / L. -1 This is most evident in the NH2-Bi-MOF group. The original digital image was converted to a grayscale image, and the ratio of grayscale values ​​(G / G0) between the sample area and the reference area was calculated. For example... Figure 12 As shown in B, in the range of 0.025-0.5g L - 1 In the NH2-Bi-MOF group, the changes before and after glyphosate addition were evident. Furthermore, it can be seen that with increasing NH2-Bi-MOF concentration, the G / G0 ratio gradually increased before and after glyphosate addition, while the difference in G / G0 before and after addition (ΔG / G0) was within 0.25 g / L. - 1 The concentration reached a maximum of 0.041 at NH2-Bi-MOF, and then decreased, a result consistent with visual observation. Therefore, 0.25 g L was selected. -1 NH2-Bi-MOF was used in subsequent experiments.

[0093] (3) Response time selection:

[0094] Under optimized paper-based material and NH2-Bi-MOF concentration conditions, the effects of adding 100 μmol L⁻¹ were investigated. -1 The change of G / G0 over time within 5 minutes after glyphosate administration, including Cu 2+ The concentration was set to 90 μmol L. -1 .

[0095] like Figure 13 As shown in Figure A, changes in the results were visible within 0-5 minutes after the addition of glyphosate, especially the gradual uniform distribution of fluorescent spots within 1 minute, after which the fluorescence intensity gradually decreased over time. Figure 13 As shown in Figure B, based on the read grayscale values, compared to the blank group, G / G0 increased to 0.60 within 1 minute, representing a signal enhancement of nearly 40%, before decreasing thereafter. This decreasing trend in fluorescence intensity is attributed to the evaporation of liquid on the test strip surface affecting the electronic transition capabilities of the MOF material, thus reducing its fluorescence properties. Therefore, the G / G0 value 1 minute after adding the glyphosate sample was selected as the signal output for accurate analysis in subsequent experiments.

[0096] Example 4: Glyphosate Detection - Construction of Standard Colorimetric Card and Standard Curve

[0097] (1) Visualized semi-quantitative detection of standard samples:

[0098] Glyphosate standards of different concentrations (1.0, 5.0, 10, 50, 100, and 200 μmol / L) were prepared. -1 The sample was processed using the glyphosate ratiometric fluorescent test strip detection method described in Example 2 and the optimized detection conditions described in Example 3. Figure 14 As shown, the fluorescence intensity in the sample area gradually increases with increasing glyphosate concentration. Based on this, a standard colorimetric card was developed for the semi-quantitative detection of glyphosate using visual visualization. The standard colorimetric card is set with five concentration reference values, where Nd represents undetectable (below 1 μmol / L). -1 ), Tr represents trace amounts (1-5 μmol L). -1 Lo is a small amount (5-10 μmol L). -1 Md is moderate (10-100 μmol L). -1 Hi is a large amount (more than 100 μmol L). -1 ).

[0099] (2) Constructing a ratio-based quantitative detection standard curve:

[0100] Since the fluorescence signal in the reference region remained stable, the grayscale ratio (G / G0) of the sample region to the reference region also increased with increasing glyphosate concentration. The test strip after glyphosate addition for 1 minute was photographed under a 365nm UV lamp, and grayscale analysis was performed using ImageJ. G / G0 was used as the analytical signal for glyphosate and correlated with the concentration of the glyphosate standard. The relationship between G / G0 and glyphosate concentration conformed to a logistic function model, and the coefficient of determination (R²) was [value missing]. 2 The value is 0.9984, such as Figure 15As shown, this test strip can be used for the quantitative detection of glyphosate. Using this test strip, the detection limit for glyphosate is 0.82 μmol / L. -1 .

[0101] Example 5: Method Adaptability: Detection of Glyphosate in Soybeans, Wheat, and Drinking Water

[0102] Glyphosate in soybeans, wheat, and drinking water was detected using the fluorescence sensor detection method in Example 1 and the ratiometric fluorescence test strip detection method in Example 2, respectively, with 5 μmol / L added to each sample. -1 and 10 μmol L -1 The recovery rate of glyphosate standard solution was calculated, and each sample was measured 5 times.

[0103] The test results are shown in Table 1. According to the results of the ratiometric fluorescent test strip, soybeans contain 4.40 μmol L⁻¹. -1 Glyphosate was detected in the other two samples, but no residual glyphosate was found. Similarly, according to the fluorescence sensor detection results, soybeans contained 4.81 μmol L⁻¹. -1 Glyphosate was detected in the other two samples, but no residual glyphosate was found in the other two samples.

[0104] Furthermore, for spiked samples, the calculated recoveries ranged from 97.6% to 109.9%, and these results were consistent with those obtained using solution sensors and LC-MS methods. This method is low-cost, simple to operate, and rapid, requiring no large instruments, and exhibits high accuracy and stability, making it a promising candidate for field monitoring of glyphosate. See Table 1 for details.

[0105] Table 1. Concentration of glyphosate in actual samples (μmol / L) -1 Recovery rate (%) and precision (%, n=5)

[0106]

[0107] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for rapid detection of glyphosate using a fluorescent sensor, characterized in that, The method includes the following steps: (1) The fluorescent MOF material NH2-Bi-MOF was dispersed in water and the pH was adjusted to weakly acidic or neutral. Cu was then added. 2+ NH2-Bi-MOF / Cu 2+ Sensing system; (2) Add glyphosate standard samples with a certain concentration gradient into the sensing system, use the ratio of fluorescence intensity before and after adding standard samples as the signal output, and linearly correlate it with glyphosate concentration to obtain a quantitative detection model; (3) The sample to be tested is added to the sensing system, and the ratio of fluorescence intensity before and after the sample to be tested is used as the signal output. By referring to the quantitative detection model, the corresponding glyphosate concentration can be obtained. The concentration of NH2-Bi-MOF in the sensing system is 0.025-0.5 g / L. -1 Cu in the sensing system 2+ Concentration of 80-100 µmol L -1 The pH of the sensing system is 5.0-7.0, and the fluorescence intensity of the sensing system after adding the sample for 0-5 minutes is used as the signal output.

2. The method according to claim 1, characterized in that, The concentration of NH2-Bi-MOF in the sensing system is 0.25 g / L. -1 .

3. The method according to claim 1, characterized in that, Cu in the sensing system 2+ Concentration of 90 µmol L -1 .

4. The method according to claim 1, characterized in that, The pH in the sensing system is 6.

0.

5. The method according to claim 1, characterized in that, The fluorescence intensity of the sensing system 1 min after adding the sample is used as the signal output.

6. The method according to claim 1, characterized in that, The fluorescent MOF material NH2-Bi-MOF was synthesized by hydrothermal method.

7. The method according to claim 1, characterized in that, The detection limit for glyphosate is 0.05 µmol / L. -1 .

8. A ratiometric fluorescent test strip for detecting glyphosate, characterized in that, The test strip comprises a paper base material, a fluorescent ring sticker, and a probe material. The fluorescent ring sticker is disposed on the paper base material as a ring-shaped reference region, and the resulting central circular region is the central sample region. The central sample region is pre-added with NH2-Bi-MOF / Cu. 2+ As a probe material, after drying, it forms an NH2-Bi-MOF / Cu structure. 2+ Ratio-type fluorescent test strips for the reaction system; The concentration of NH2-Bi-MOF in the reaction system is 0.025-1.0 g / L. -1 Cu in the reaction system 2+ Concentration of 80-100 µmol L -1 NH2-Bi-MOF / Cu added dropwise to the central sample area 2+ The reaction system volume is 1-5 μL.

9. The ratio-type fluorescent test strip according to claim 8, characterized in that, The paper-based material includes any one of glass fiber membrane, Whatman No.1 paper filter, and cellulose acetate membrane. The fluorescent ring sticker is adhered to the paper-based material. The paper-based material is square, and its side length is greater than or equal to the outer diameter of the fluorescent ring sticker. The ratio of the outer diameter to the inner diameter of the fluorescent ring sticker is (4-10):

3.

10. The ratio-type fluorescent test strip according to claim 9, characterized in that, The ratio of the outer diameter to the inner diameter of the fluorescent ring sticker is 5:

3.

11. The ratiometric fluorescent test strip according to claim 8, characterized in that, The preparation of the test strip includes the following process: (1) Cut paper-based materials such as glass fiber membrane to obtain square test paper pieces, punch holes in adhesive fluorescent roll paper to obtain fluorescent ring stickers, and assemble the fluorescent ring stickers on top of the glass fiber membrane test paper. (2) The fluorescent MOF material NH2-Bi-MOF was synthesized by hydrothermal reaction, and Cu was added in advance. 2+ NH2-Bi-MOF / Cu 2+ The reaction system was added dropwise to the sample area of ​​the test strip, and after drying, a ratiometric fluorescent test strip was obtained.

12. The ratio-type fluorescent test strip according to claim 8, characterized in that, The concentration of NH2-Bi-MOF in the reaction system is 0.025-0.5 g / L. -1 .

13. The ratiometric fluorescent test strip according to claim 12, characterized in that, The concentration of NH2-Bi-MOF in the reaction system was 0.25 g / L. -1 .

14. The ratio-type fluorescent test strip according to claim 8, characterized in that, Cu in the reaction system 2+ Concentration of 90 µmol L -1 .

15. The ratiometric fluorescent test strip according to claim 8, characterized in that, NH2-Bi-MOF / Cu added dropwise to the central sample area 2+ The reaction system volume is 2 μL.

16. The ratio-type fluorescent test strip according to claim 8, characterized in that, The detection limit for glyphosate on the ratiometric fluorescent test strip is 0.82 µmol / L. -1 .

17. A method for detecting glyphosate using ratiometric fluorescent test strips, characterized in that, Includes the following steps: (1) The sample to be tested is dropped into the central sample area of ​​the ratiometric fluorescent test strip as described in any one of claims 8-16; (2) After the reaction, the glyphosate concentration was semi-quantitatively analyzed by comparing it with the standard colorimetric card under ultraviolet light irradiation, and / or by taking pictures under ultraviolet light irradiation to obtain image information and perform grayscale analysis. The grayscale values ​​of the central sample area and the annular reference area were compared, and the glyphosate concentration was quantitatively analyzed according to the ratio-type quantitative detection model. The standard colorimetric card is a standard concentration reference model constructed by linearly correlating the results of standard samples with known concentration gradients with glyphosate concentrations. The ratiometric quantitative detection model is a standard curve constructed by linearly correlating the results of standard samples with known concentration gradients with glyphosate concentration.

18. The method according to claim 17, characterized in that, In step (1), the volume of the sample to be tested added to the central sample area is 1-5 μL; In step (2), the wavelength of the ultraviolet lamp irradiation is 365 nm, and the grayscale analysis is performed using ImageJ software; The standard colorimetric card has five concentration reference values, where Nd is not detected: below 1 µmol / L. -1 Tr represents trace amounts: 1~5 µmol L -1 Lo is in small amounts: 5–10 µmol L -1 Md is moderate: 10–100 µmol L -1 Hi is in large quantities: exceeding 100 µmol L -1 ; After adding the sample to be tested for 0-5 minutes, the test strip is photographed and analyzed using a mobile phone or camera.

19. A detection device, characterized in that, The detection device includes a ratiometric fluorescent test strip as described in any one of claims 8-16, and the form of the detection device includes a reagent kit.

20. The application of the ratiometric fluorescent test strip as described in any one of claims 8-16, or the detection device as described in claim 19, or the method as described in any one of claims 1-7, or the method as described in claim 17 or 18, in the detection of glyphosate in water, soil, and food.

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