Near-ultraviolet emitting copper nanoclusters and preparation method and application thereof

Abstract

This invention discloses a near-ultraviolet emitting copper nanocluster, its preparation method, and its applications. The chemical composition of the copper nanocluster is Cu4MPD7, and its fluorescence emission peak is located near 343 nm. The preparation method employs a simple one-pot aqueous phase method, using m-phenylenediamine as a reducing agent and ligand, reacting it with copper sulfate at room temperature followed by centrifugation. This nanocluster can be used as a fluorescent probe, emitting near-ultraviolet emission through its surface Cu... 2+ The specific binding with glyphosate induces fluorescence enhancement, enabling highly sensitive and selective detection of glyphosate, which has been successfully applied to the analysis of actual samples. Furthermore, due to its near-ultraviolet emission properties and excellent biocompatibility, this nanocluster also has significant application potential in fields such as biolabeling, in vivo imaging, disease treatment, catalytic materials, and the preparation of composite nanomaterials. This invention features a simple process, low cost, and environmental friendliness, providing new materials and methods for pesticide residue detection and the development of functional nanomaterials.

Description

Technical Field

[0001] This invention belongs to the field of functional nanomaterials and analytical sensing technology, specifically relating to a near-ultraviolet emitting copper nanocluster, its preparation method, and its application. Background Technology

[0002] Glyphosate (C3H8NO5P, N-(Phosphonomethyl)glycine, NPmg) is currently the most widely used broad-spectrum herbicide globally, and is applied extensively in agricultural production. However, its high water solubility, potential biotoxicity, and carcinogenic risks have posed a serious threat to the ecological environment and human health. Various countries have established strict limits for glyphosate residues in food and the environment. For example, China sets the maximum residue limit for tea at 1.0 mg / kg, while the European Union sets the limit for glyphosate in drinking water as low as 0.1 µg / L. Therefore, developing highly sensitive, selective, rapid, and economical methods for detecting glyphosate is of significant practical importance.

[0003] However, the molecular characteristics of glyphosate make it one of the most difficult pesticide residues to detect. Firstly, glyphosate is a highly polar, water-soluble small organic molecule, making it difficult to extract from the aqueous phase using conventional organic solvents. This results in weak or no retention on traditional reversed-phase chromatography columns (C18 columns), making direct analysis impossible. Secondly, the glyphosate molecule itself lacks natural chromophores and fluorophores, rendering it undetectable by conventional high-performance liquid chromatography-UV / fluorescence detectors, requiring complex derivatization. Finally, glyphosate molecules readily bind to metal ions and soil matrices, affecting extraction efficiency and consequently impacting detection accuracy.

[0004] Currently, glyphosate detection mainly relies on chromatography-mass spectrometry (GC-MS) and immunoassay. While these methods offer high accuracy, they generally suffer from drawbacks such as expensive equipment, complex sample preparation, long processing times, and reliance on specialized personnel, making them unsuitable for rapid on-site screening and real-time monitoring. In recent years, fluorescence sensing-based detection methods have gained widespread attention due to their ease of operation, rapid response, and high sensitivity. However, glyphosate molecules themselves lack fluorophores or chromophores and are highly polar, readily binding to matrices, making direct fluorescence detection extremely challenging.

[0005] Metal nanoclusters (such as Au, Ag, Pt, and Cu) are novel fluorescent nanomaterials that have shown great potential in fields such as biosensing, cell labeling, in vivo imaging, and food and environmental analysis due to their advantages such as small size, tunable fluorescence, and good biocompatibility. Among them, copper nanoclusters (CuNCs) are considered one of the most promising fluorescent probes due to their low cost, abundant sources, and environmental friendliness. Existing research has attempted to use them for glyphosate detection; for example, Chinese patent application CN113866138A discloses a competitive fluorescence detection method based on DNA-templated silver nanoclusters and copper ions. Although this method has high sensitivity, it still has the following limitations: (1) It relies on DNA template and external copper ions for mediation, the system is complex and the cost is high; (2) The probe synthesis steps are complicated and require strict conditions such as low temperature incubation and light-avoidance reaction; (3) The emission of the silver nanoclusters used is located in the visible light region (about 620 nm), which is easily affected by the autofluorescence of the sample matrix; (4) The detection mechanism is of the "fluorescence quenching and recovery" type, the signal change direction is single, and it is easily affected by environmental interference.

[0006] In recent years, a new sensing and detection method based on nanomaterials has emerged: the nanozyme sensing strategy based on catalytic activity inhibition. This type of method utilizes the binding of glyphosate to the active center of nanozymes (usually copper-based materials), inhibiting their enzyme-like (e.g., POD-like peroxidase) activity, thereby affecting the colorimetric or chemiluminescent reaction, and detecting the glyphosate through changes in absorbance or luminescence intensity. For example, materials such as copper single-atom coordinated amino covalent organic frameworks (Chinese patent application CN120741375A), copper-carbon quantum dots (Chinese patent application CN119186562A), Cu-CDs / ABEI@Ag composite nanozymes (Chinese patent application CN121231459A), and copper-based nanozymes with a network structure (Chinese patent application CN120286074A) have all demonstrated the ability to detect glyphosate. These nanozyme methods generally exhibit good sensitivity and a certain degree of selectivity, but their detection mechanisms rely on the addition of exogenous substrates (such as TMB and H2O2) and complex colorimetric / luminescent reaction systems, increasing the number of operational steps and detection costs. Furthermore, the synthesis of nanozymes often involves high-temperature, high-pressure hydrothermal, solvothermal, or complex post-modification processes (such as the multi-step synthesis of the framework and copper loading required in Chinese patent application CN120741375A), making the process less simple, and the catalytic activity is easily affected by environmental factors such as pH and temperature.

[0007] Currently, in the field of fluorescent nanomaterials such as copper nanoclusters (CuNCs), existing research mainly focuses on CuNCs that emit in the visible light region. Research on the preparation of near-ultraviolet emitting CuNCs and their direct application in glyphosate detection remains relatively scarce. Existing near-ultraviolet emitting CuNCs often involve complex synthesis steps, low quantum yield, and poor stability, limiting their application in trace analysis. Therefore, developing a copper nanocluster probe that is easy to synthesize, emits near-ultraviolet light, has good stability, and can directly exhibit a specific fluorescence response to glyphosate is of great significance for advancing the development of rapid glyphosate detection technology. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a near-ultraviolet emitting copper nanoclusters, their preparation method, and applications. A very simple room-temperature aqueous one-pot method is used to synthesize uniformly dispersed CuNCs, whose surface Cu... 2+ It can specifically bind to glyphosate, leading to a significant enhancement of fluorescence, thus enabling direct and rapid detection of glyphosate without the need for external reagents, with simple operation, high sensitivity, and high selectivity. It effectively expands the emission range of traditional CuNCs and compensates for the spectral limitations of CuNCs in the near-ultraviolet region, which is beneficial for expanding the application range of CuNCs in environmental and food analysis and detection. It solves the technical challenge of the short-wavelength emission limitation of CuNCs, while simultaneously achieving highly selective and sensitive detection of small-molecule pesticides.

[0009] This invention is achieved through the following technical solution:

[0010] A method for preparing near-ultraviolet emitting copper nanoclusters includes the following steps:

[0011] Step 1) Mix the aqueous solution of m-phenylenediamine with the aqueous solution of copper sulfate and stir at 1500~2500 rpm for 2~5 min;

[0012] Step 2) Add purified water to the solution obtained in Step 1) and continue stirring at 1500~2500 rpm for 0.5~2 min;

[0013] Step 3) Centrifuge the solution obtained in Step 2) at 5000~8000 rpm for 3~10 min to remove the precipitate and collect the supernatant.

[0014] Preferably, the concentration of the m-phenylenediamine aqueous solution in step 1) is 50~100 mM, the concentration of the copper sulfate aqueous solution is 0.08~0.15 M, and the volume ratio of m-phenylenediamine to copper sulfate is 4:1~6:1.

[0015] A near-ultraviolet emitting copper nanocluster was prepared by the above-described method; the chemical composition of the copper nanocluster is Cu4MPD7, and its maximum fluorescence emission wavelength is around 343 nm.

[0016] The above-mentioned near-ultraviolet emitting copper nanoclusters are used in the detection of glyphosate.

[0017] Preferably, it includes the following steps:

[0018] The near-ultraviolet emitting copper nanoclusters were mixed with HEPES buffer, and then the sample to be tested or glyphosate standard solution was added. The mixture was allowed to stand for 10-20 min. The fluorescence intensity change was measured at an excitation wavelength of 260-280 nm, and the glyphosate concentration was calculated based on the degree of fluorescence enhancement.

[0019] Preferably, the pH value of the HEPES buffer is 7.0~7.8, the concentration is 40~60 mM, the total volume of the reaction system is 400~600 μL, and the amount of near-ultraviolet emitting copper nanoclusters added is 5~15 μL.

[0020] The above-mentioned near-ultraviolet emitting copper nanoclusters are used in the preparation of formulations for biolabeling or in vivo imaging.

[0021] The above-mentioned near-ultraviolet emitting copper nanoclusters are used in the preparation of catalytic materials or composite nanomaterials.

[0022] A glyphosate detection kit includes the aforementioned near-ultraviolet emitting copper nanoclusters, HEPES buffer, and instructions for use.

[0023] A composite nanomaterial comprising the aforementioned near-ultraviolet emitting copper nanoclusters as a functional component.

[0024] The beneficial effects of this invention are as follows:

[0025] (1) This invention employs a "one-pot" aqueous phase stirring technique, which only requires simple mixing and stirring of m-phenylenediamine (MPD) and copper sulfate (CuSO4) solution at room temperature, followed by centrifugation to obtain the target copper nanoclusters (CuNCs). The entire process does not require high temperature and high pressure, inert gas protection, templates or expensive reagents, nor does it require complex post-processing steps. The conditions are mild, the operation is simple, the time consumption is short, and the energy consumption is low, making it very suitable for large-scale preparation and possessing significant industrial application potential and cost advantages.

[0026] (2) The CuNCs prepared in this invention are rich in divalent copper ions (Cu) on their surface. 2+This substance can specifically bind directly to glyphosate molecules, leading to a significant enhancement of the near-ultraviolet fluorescence signal of CuNCs themselves. This "signal-on" detection mode requires no addition of any exogenous catalysts, chromogenic substrates, oxidants, or additional metal ions, simplifying the operation process, reducing potential interference factors, and making detection more convenient, faster, and more reliable.

[0027] (3) The present invention successfully prepared near-ultraviolet emitting CuNCs with a definite chemical composition (Cu4MPD7), and the maximum fluorescence emission peak is located at about 343 nm. This near-ultraviolet emission characteristic effectively avoids the autofluorescence background common in the visible light region of most complex sample matrices (such as food and environmental water bodies), significantly improves the signal-to-noise ratio and anti-interference ability of detection, and provides favorable conditions for the accurate quantification of trace targets in complex systems.

[0028] (4) The CuNCs probe of this invention exhibits excellent detection performance for glyphosate. Under optimized conditions, it achieves sensitive detection with a wide linear range and low detection limit. Simultaneously, the probe is also effective against various common coexisting ions (such as K+). + Na + Ca 2+ It exhibits good anti-interference ability with other structurally similar pesticide molecules (such as diuron and chlorpyrifos), showing a high degree of selective recognition of glyphosate molecules.

[0029] (5) The detection method constructed in this invention has been successfully applied to the determination of glyphosate in actual samples (such as millet, soybeans, and pond water). The spiked recovery experiment results showed that the recovery rate was ideal (97.52%~105.07%) and the relative standard deviation was small (RSD<3%), proving that the method has excellent accuracy, reliability, and practicality in actual complex matrices. In addition, the near-ultraviolet emitting CuNCs of this invention can not only be used for the detection of pesticide molecules, but also have broad application potential in fields such as biolabeling, in vivo imaging, catalysis, and composite material preparation due to their unique optical properties and good biocompatibility. Attached Figure Description

[0030] Figure 1 The morphology and composition of CuNCs in Example 1 are shown in Figure A: Transmission electron microscopy (TEM) image (scale 200 nm), inset image is high-resolution TEM image (scale 5 nm); B: Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) image.

[0031] Figure 2 The surface chemical states of CuNCs in Example 1 are as follows: A is the Fourier Transmission Infrared Spectrum (FT-IR); B is the full spectrum of X-ray photoelectron spectroscopy (XPS); C is the high-resolution Cu 2p spectrum; D is the high-resolution N 1s spectrum.

[0032] Figure 3 Optical properties of CuNCs in Example 1: A shows the UV-Vis absorption, excitation, and emission spectra; B shows the fluorescence emission spectra under different excitations.

[0033] Figure 4 The changes in the valence state of copper ions on the surface of the CuNCs fluorescent probe before and after the addition of glyphosate in Example 2;

[0034] Figure 5 Example 2 shows the detection of glyphosate using CuNCs fluorescent probes: A is the change curve of CuNCs fluorescence intensity after adding different concentrations of glyphosate; B is the standard working curve.

[0035] Figure 6 This shows the fluorescence response of the CuNCs fluorescent probe in Example 3 in the presence of various competing ions or molecules. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0037] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] Example 1: Preparation and characterization of near-ultraviolet emitting copper nanoclusters (CuNCs)

[0040] 1. Preparation of near-ultraviolet emitting CuNCs using aqueous phase stirring technique

[0041] A method for preparing near-ultraviolet emitting copper nanoclusters, the specific steps of which are as follows:

[0042] (1) Prepare an aqueous solution of 75 mM m-phenylenediamine (C6H8N2, MPD) and an aqueous solution of 0.1 M copper sulfate (CuSO4).

[0043] (2) Measure 500 µL of MPD aqueous solution and add it to a clean centrifuge tube.

[0044] (3) Add 100 µL of CuSO4 aqueous solution to the centrifuge tubes.

[0045] (4) Immediately place the centrifuge tube on a vortex mixer and stir continuously at a speed of about 2000 rpm for 3 minutes. Observe the color change of the solution.

[0046] (5) Then, add 500 µL of ultrapure water to the above reaction solution and stir continuously for 1 min at a speed of about 2000 rpm using a vortex mixer.

[0047] (6) Centrifuge the stirred solution at 6000 rpm for 5 min to remove the precipitate and collect the supernatant, which is the CuNCs stock solution (concentration of approximately 0.87 mg / mL).

[0048] (7) Store the CuNCs stock solution in a 4°C refrigerator for later use.

[0049] 2. Characterization Results and Analysis

[0050] (1) Morphology and composition

[0051] TEM characterization ( Figure 1 (A) shows that the prepared CuNCs exhibit a short rod-like shape. High-resolution TEM (…) Figure 1 (Inset A) further shows that the rod-like structure is assembled from four cluster units. MALDI-TOF-MS analysis ( Figure 1 (B) Confirms that the precise composition of each cluster unit is Cu4MPD7, which contains 4 Cu atoms and 7 MPD ligands.

[0052] (2) Surface chemical state

[0053] FT-IR spectroscopy ( Figure 2 As shown in Figure A), CuNCs exhibited better performance at 615 cm⁻¹ compared to pure MPD ligands. -1 A new characteristic peak appeared, which is attributed to the vibration of the Cu-N bond, confirming that the MPD ligand is anchored to the nanocluster surface through Cu-N bonds, and the migration of the CN bond absorption band also proves this point; at the same time, the absorption bands are at 3400, 3000 and 1400~1650 cm⁻¹. -1 The retention of [the structure] indicates that MPD maintains its basic structure on the copper cluster surface. XPS full spectrum ( Figure 2 (B) Confirms that CuNCs contain Cu, C, N, and O elements, with C and N belonging to MPD ligands and O belonging to surface H₂O. High-resolution Cu 2p spectrum ( Figure 2 The fitting results of the C1s spectrum showed that the Cu core simultaneously contained three valence states: Cu(0), Cu(I), and Cu(II). The presence of surface Cu(II) was confirmed as a necessary and sufficient condition for subsequent detection of glyphosate. High-resolution N1s spectra (…) Figure 2 The Cu-N peak appearing in (D) further supports the conclusions of FT-IR.

[0054] (3) Optical properties

[0055] UV-Vis absorption spectroscopy shows that the UV absorption spectrum of the pure MPD ligand is different. Figure 3 Compared to curve a), CuNCs (A, curve a) Figure 3 In curve A (and curve b), while retaining the absorption peak of the MPD ligand, a distinct broad absorption band appears near 480 nm, which is a characteristic absorption peak of metal nanoclusters. Fluorescence spectroscopy (…) Figure 3 As shown in curve A (d), the maximum fluorescence emission wavelength of the prepared CuNCs is located in the near-ultraviolet region at ~343 nm. Excitation spectrum ( Figure 3 (B) The optimal excitation wavelength for CuNCs was determined to be 270 nm, which coincides with the excitation wavelength of CuNCs. Figure 3 (A in the middle, curve c). These data together demonstrate that CuNCs with near-ultraviolet emission properties have been successfully prepared.

[0056] Example 2: Detection of glyphosate (NPmg) using near-ultraviolet emission CuNCs fluorescent probes

[0057] 1. Drawing the standard curve

[0058] (1) Take a series of 2.0 mL centrifuge tubes and label them.

[0059] (2) Add 10.0 µL of CuNCs stock solution prepared in Example 1 (approximately 0.87 mg / mL) to each centrifuge tube.

[0060] (3) Add 50 µL of HEPES buffer solution (pH 7.4, 50 mM) to each tube in sequence.

[0061] (4) Add NPmg standard solution of different concentrations (0, 0.01, 0.1, 0.5, 1, 5, 10, 20, 30, 50, 100, 150, 200, 300 µM) to each tube respectively.

[0062] (5) After gently vortexing and mixing, let it stand at room temperature for 1 min for incubation.

[0063] (6) Use ultrapure water to bring the volume of the entire reaction system to 500 µL.

[0064] (7) Let the reaction stand at room temperature for 20 min.

[0065] (8) Detection was performed using a fluorescence spectrophotometer. The excitation wavelength was set to 270 nm, and the excitation and emission slit widths were 5 nm and 3 nm, respectively. The fluorescence intensity (FL) of CuNCs in each tube at the maximum emission wavelength (approximately 343 nm) was scanned and recorded. Figure 5 As shown in Figure A.

[0066] (9) Taking the fluorescence intensity of the sample without NPmg as FL0, calculate the fluorescence intensity change value at each concentration: (FL-FL0) / FL0.

[0067] (10) Plot a standard curve with NP mg concentration (x, µM) as the abscissa and fluorescence intensity change [(FL-FL0) / FL0] (y) as the ordinate, and obtain the linear regression equation, as follows: Figure 5 As shown in B.

[0068] 2. Performance Evaluation

[0069] (1) Detection mechanism verification

[0070] Given the specific chelation effect between free Cu(II) and NPmg, this example analyzes the Cu LMM Auger peak before and after the addition of NPmg. Figure 4 As shown, the amount of Cu(II) on the CuNCs surface decreased sharply after the addition of NPmg, with the Cu(II) / Cu (total) ratio dropping from 0.34 to 0.05. This indicates that the presence of NPmg can transform Cu(II) into Cu(I) / Cu(O). That is, when NPmg is added to a CuNCs probe solution containing a large amount of Cu(II) on its surface, the NPmg containing O / N electron-donating groups will coordinate with the exposed Cu(II) sites on the surface through electron acceptor-donor chelation, forming a stable interfacial electronic binding state on the CuNCs surface. This effectively suppresses nonradiative transitions caused by Cu(II) surface defects, ultimately enhancing exciton luminescence. This specific chelation reduces valence defects and nonradiative transitions on the CuNCs surface, resulting in a significant enhancement of its fluorescence emission (“Turn-on” signal).

[0071] (2) Performance results

[0072] Under optimized conditions (pH 7.4 HEPES buffer, reaction time 20 min, excitation at 270 nm, emission at 343 nm), the fluorescence intensity of the CuNCs probe was measured at different concentration gradients (0–300 µM) of glyphosate. The results are as follows: Figure 5 As shown in Figure A, the fluorescence intensity increases significantly with increasing glyphosate concentration.

[0073] Standard curve such as Figure 5 As shown in Figure B, the signal response exhibits an excellent linear relationship with concentration within the range of 0.01 µM to 200 µM. The linear regression equation is: y = 0.0099x + 0.0757, and the correlation coefficient R0 is [value missing]. 2 =0.9978. Based on this linear equation, and using a signal-to-noise ratio of 3 (3σ / k), the limit of detection (LOD) for glyphosate using this method is less than 0.1 µM.

[0074] The method in this embodiment is based on a direct fluorescence enhancement effect, which achieves wide linear range and high sensitivity detection of glyphosate without signal conversion or amplification steps.

[0075] Example 3: Study on the Selectivity of Sensors

[0076] 1. Experimental Procedure

[0077] (1) Take a series of 2.0 mL centrifuge tubes, the number of which is consistent with the number of competing substances to be tested. The competing substances include a series of competing metal cations (Zn). 2+ Mg 2+ Ca 2+ K + Na + Fe 2+ Mn 2+ Co 2+ The test included competing pesticides / small biological molecules (diuron, carbaryl, carbendazim, paraquat, chlorpyrifos, glucose, ascorbic acid), and a separate tube served as a blank control (containing only buffer and CuNCs).

[0078] (2) Add 10.0 µL of CuNCs stock solution (approximately 0.87 mg / mL) prepared in Example 1 and 50 µL of HEPES buffer solution (pH 7.4, 50 mM) to each tube.

[0079] (3) Except for the blank control tube, each of the other tubes was given an equal amount of a single competitor solution with a concentration of 300 µM to examine its interference ability.

[0080] (4) After gently vortexing and incubating for 1 min, bring the volume up to 500 µL with ultrapure water.

[0081] (5) Let the reaction stand at room temperature for 20 min.

[0082] (6) Under the same fluorescence detection conditions as in Example 2, the fluorescence intensity of CuNCs in each tube was measured.

[0083] (7) Calculate the fluorescence intensity change rate in the presence of each competitor: (FL) 竞争物 -FL0) / FL0.

[0084] (8) Plot a bar chart (selectivity trend chart) with the types of competing substances as the x-axis and the rate of change in fluorescence intensity as the y-axis, such as... Figure 6 As shown in the figure. By comparing the significant fluorescence enhancement caused by glyphosate with the weak changes caused by other competitors, the specificity of this sensor for glyphosate was verified.

[0085] 2. Experimental Results and Analysis

[0086] Experimental results are as follows Figure 6 As shown, the fluorescence intensity change rates caused by each interfering substance are compared with the significant enhancement caused by glyphosate. Only glyphosate can induce a strong fluorescence "on" signal, while the effects of all tested metal ions and other organic molecules on the fluorescence of CuNCs are negligible, with change rates far lower than the response value of glyphosate.

[0087] The experimental results of this embodiment strongly demonstrate that the sensor has excellent selectivity for glyphosate. This selectivity stems from the unique and strong chelating ability between the phosphonic acid and carboxyl functional groups in the glyphosate molecule and specific Cu(II) sites on the CuNCs surface. This interaction is far stronger than the interaction between other ions or molecules and the probe.

[0088] Example 4: Actual Sample Testing (Spike Recovery Experiment)

[0089] 1. Sample pretreatment

[0090] (1) Millet / Soybean

[0091] Weigh 1.0 g of the pulverized sample, add 20 mL of ultrapure water, mix well, then add 10 mL of dichloromethane and sonicate for 15 min. Centrifuge at 4500 rpm for 10 min, collect the supernatant, and filter through a 0.22 µm filter membrane. Evaporate and concentrate the filtrate to approximately 5.0 mL under a gentle nitrogen stream to obtain the millet / soybean extract, which is ready for use.

[0092] (2) Pond water

[0093] The collected pond water samples were first filtered through a 0.45 µm filter membrane and then centrifuged at 10,000 rpm for 15 min to remove potential large particulate impurities. The supernatant was then diluted 50 times with 5 mM HEPES buffer (pH 7.4) to obtain a diluted pond water solution for later use.

[0094] 2. Spikes and Testing

[0095] (1) The actual sample solution after treatment (millet extract, soybean extract, pond water dilution) was used as the matrix.

[0096] (2) Take several equal portions of sample matrix and add glyphosate standard solutions of different concentrations (10, 50, 100 μM) to them for spiking.

[0097] (3) Three parallel experiments (n=3) were conducted at each concentration level. The spiked sample solution was used instead of the glyphosate standard solution for operation and fluorescence detection, following the method in Example 2.

[0098] (4) Based on the measured change in fluorescence intensity, substitute it into the linear equation of the standard curve obtained in Example 2 to calculate the concentration of glyphosate in the spiked sample.

[0099] (5) Calculate the recovery rate and the relative standard deviation (RSD) of parallel samples to evaluate the accuracy and precision of the method.

[0100]

[0101] 3. Experimental Results and Analysis

[0102] Table 1. Analytical results of glyphosate in actual samples (±SD, n=3)

[0103]

[0104] As shown in Table 1, the recoveries of glyphosate in all actual samples were within the ideal range of 97.52% to 105.07%, and the RSDs of all parallel experiments were less than 3%. This fully demonstrates that the near-ultraviolet emission CuNCs-based fluorescence sensing platform constructed in this invention can effectively overcome the interference of complex matrices in actual applications, enabling accurate, reliable, and highly precise quantitative analysis of glyphosate in food and environmental samples, and possesses significant practical application value.

[0105] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for preparing near-ultraviolet emitting copper nanoclusters, characterized in that, Includes the following steps: Step 1) Mix the aqueous solution of m-phenylenediamine with the aqueous solution of copper sulfate and stir at 1500~2500 rpm for 2~5 min; Step 2) Add purified water to the solution obtained in Step 1) and continue stirring at 1500~2500 rpm for 0.5~2 min; Step 3) Centrifuge the solution obtained in Step 2) at 5000~8000 rpm for 3~10 min to remove the precipitate and collect the supernatant.

2. The method for preparing near-ultraviolet emitting copper nanoclusters according to claim 1, characterized in that, Step 1) The concentration of the m-phenylenediamine aqueous solution is 50~100 mM, and the concentration of the copper sulfate aqueous solution is 0.08~0.15 M; the volume ratio of m-phenylenediamine to copper sulfate is 4:1~6:

1.

3. A near-ultraviolet emitting copper nanocluster, characterized in that, The copper nanoclusters are prepared by the preparation method described in claim 1 or 2; the chemical composition of the copper nanoclusters is Cu4MPD7, and the maximum fluorescence emission wavelength is located near 343 nm.

4. The application of near-ultraviolet emitting copper nanoclusters as described in claim 3 in the detection of glyphosate.

5. The application according to claim 4, characterized in that, Includes the following steps: The near-ultraviolet emitting copper nanoclusters were mixed with HEPES buffer, and then the sample to be tested or glyphosate standard solution was added. The mixture was allowed to stand for 10-20 min. The fluorescence intensity change was measured at an excitation wavelength of 260-280 nm, and the glyphosate concentration was calculated based on the degree of fluorescence enhancement.

6. The application according to claim 5, characterized in that, The HEPES buffer solution has a pH of 7.0-7.8 and a concentration of 40-60 mM; the total volume of the reaction system is 400-600 μL; and the amount of near-ultraviolet emitting copper nanoclusters added is 5-15 μL.

7. The use of the near-ultraviolet emitting copper nanoclusters as described in claim 4 in the preparation of formulations for biolabeling or in vivo imaging.

8. The application of the near-ultraviolet emitting copper nanoclusters as described in claim 4 in the preparation of catalytic materials or composite nanomaterials.

9. A glyphosate detection kit, characterized in that, Includes the near-ultraviolet emitting copper nanoclusters as described in claim 4, HEPES buffer, and instructions for use.

10. A composite nanomaterial, characterized in that, It includes the near-ultraviolet emitting copper nanoclusters as described in claim 4 as a functional component.

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