Bicolor fluorescent probe for detecting methyl orange and preparation method and application thereof

Abstract

This invention relates to the field of fluorescence detection technology, specifically disclosing a dual-colorimetric fluorescent probe for detecting methyl orange, its preparation method, and its application. The probe is prepared by in-situ synthesis of carbon dots and copper nanoclusters. The carbon dots are synthesized from Bermuda grass as a precursor via a hydrothermal method, and the copper nanoclusters are obtained by reducing copper ions with penicillamine and bovine serum albumin as dual stabilizers. The dual-colorimetric fluorescent probe provided by this invention has a detection limit for methyl orange as low as 0.06 μM, exhibiting good selectivity, stability, and anti-interference ability. It can be applied to the spiked recovery detection of methyl orange in actual water samples and can achieve visualized semi-quantitative analysis through filter paper loading, making it suitable for rapid on-site screening.

Description

Technical Field

[0001] This invention relates to the field of fluorescence detection technology, specifically to a dual-colorimetric fluorescent probe for detecting methyl orange, its preparation method, and its application. Background Technology

[0002] Methyl orange (MO) is a typical azo synthetic dye. Due to its stable dyeing properties and low cost, it is often used as an acid-base indicator in scientific research and is also widely used in industries such as textiles, dyeing, printing, and papermaking. However, numerous studies have confirmed that MO is difficult to degrade in the environment and is toxic. Long-term persistence of MO in aquatic environments can damage aquatic ecosystems and lead to eutrophication. More importantly, MO can enter the human body through the food chain, posing potential harm to organs such as the liver and kidneys, and may even indirectly cause cancer.

[0003] Currently, the main methods for detecting MO include high-performance liquid chromatography (HPLC), ultraviolet-visible spectrophotometry (UV-Vis spectrophotometry), and fluorescence detection. Among these, fluorescence detection is the most commonly used due to its simplicity and fast response. However, existing fluorescent probes have the following problems: their physicochemical properties are unstable and easily affected by temperature and pH; their recognition of MO is not specific enough, and they are easily interfered with by other substances in complex water samples, resulting in the sensitivity and accuracy of detection failing to meet practical needs and making it impossible to accurately measure low concentrations of MO residues. Summary of the Invention

[0004] To detect low concentrations of methyl orange residues, this invention provides a dual-colorimetric fluorescent probe for detecting methyl orange, its preparation method, and its application. The dual-colorimetric fluorescent probe provided by this invention has a detection limit for methyl orange as low as 0.06 μM, exhibits good selectivity, stability, and anti-interference ability, and can be applied to the spiked recovery detection of methyl orange in actual water samples. Furthermore, it can achieve visualized semi-quantitative analysis through filter paper loading, making it suitable for rapid on-site screening.

[0005] This invention provides a dual-colorimetric fluorescent probe for detecting methyl orange. The dual-colorimetric fluorescent probe is a carbon dot / copper nanocluster composite probe, which is synthesized from bistable copper nanoclusters and carbon dots through an in-situ synthesis method. The bistable copper nanoclusters are prepared by reacting docosapentaenoic acid (DEA) and copper salt to form a precursor, which is then stabilized with bovine serum albumin (BSA), wherein DEA and BSA serve as dual stabilizers. The carbon dots are prepared from plant biomass via a hydrothermal method.

[0006] The CDs-Cu NCs fluorescent probe provided by this invention is synthesized from bistable copper nanoclusters and carbon dots through an in-situ synthesis method. The bistable copper nanoclusters utilize docosapentaenoic acid (DPA) and BSA as dual stabilizers, resulting in higher stability of the final copper nanoclusters. The carbon dots are prepared from readily available natural Bermuda grass roots via a hydrothermal method. Bermuda grass roots are rich in carbon and nitrogen, enabling self-doping and possessing both excellent optical properties and good biocompatibility.

[0007] Furthermore, the plant biomass is Bermuda grass root.

[0008] The present invention also provides a method for preparing the aforementioned dual-colorimetric fluorescent probe, comprising the following steps: Eicosapentaenoic acid solution was mixed with copper salt solution to form a precursor, which was then stabilized by adding bovine serum albumin solution and purified by dialysis to obtain bistable copper nanoclusters solution. Plant biomass is dried, pulverized, and sieved to obtain grass powder, which is dispersed in water and centrifuged to obtain a uniform suspension. The suspension is subjected to a hydrothermal reaction, and the reaction product is purified by centrifugation, filtration, and dialysis to obtain a carbon dot solution. A carbon dot solution was mixed with a bistable copper nanocluster solution, and after stirring and reaction, the mixture was purified by dialysis to obtain a carbon dot / copper nanocluster composite probe.

[0009] Furthermore, the carbon dot solution is mixed with the bistable copper nanocluster solution in an equal volume ratio.

[0010] The present invention also provides the application of the aforementioned dual-colorimetric fluorescent probe in the detection of methyl orange.

[0011] Furthermore, during detection, the sample to be tested is mixed with the dual-colorimetric fluorescent probe, and its fluorescence emission spectrum in the range of 350 nm to 690 nm is measured at an excitation wavelength of 345 nm. The ratio of fluorescence intensity at 638 nm to 448 nm is calculated, and the concentration of methyl orange in the sample is determined according to the established ratio-concentration standard curve.

[0012] Furthermore, the application also includes visual semi-quantitative detection: by observing the color change of the dual-color fluorescent probe after it reacts with different concentrations of methyl orange under ultraviolet light or sunlight, and comparing it with a standard color card, rapid on-site screening of methyl orange can be achieved.

[0013] Furthermore, the sample to be tested is an environmental water sample, including tap water, river water, or industrial wastewater.

[0014] Furthermore, the bistable copper nanoclusters are prepared by reacting D-penicillamine and copper salt to form a precursor, followed by stabilization treatment with bovine serum albumin; the carbon dots are prepared from plant biomass via a hydrothermal method.

[0015] Furthermore, the carbon dot / copper nanocluster composite probe can generate dual emission fluorescence signals at 448nm and 638nm under an excitation wavelength of 345nm.

[0016] The present invention also provides a method for preparing the aforementioned dual-colorimetric fluorescent probe for detecting methyl orange, comprising the following steps: D-penicillamine solution was mixed with copper salt solution to form a precursor, which was then stabilized by adding bovine serum albumin solution and purified by dialysis to obtain bis-stable copper nanoclusters solution. Bermuda grass is dried, pulverized, and sieved to obtain grass powder, which is then dispersed in water and centrifuged to obtain a uniform suspension. The suspension is subjected to a hydrothermal reaction, and the reaction product is purified by centrifugation, filtration, and dialysis to obtain a carbon dot solution. A carbon dot solution was mixed with a bistable copper nanocluster solution, and after stirring and reaction, the mixture was purified by dialysis to obtain a carbon dot / copper nanocluster composite probe.

[0017] Furthermore, the carbon dot solution is mixed with the bistable copper nanocluster solution in an equal volume ratio.

[0018] The present invention also provides the application of the aforementioned dual-colorimetric fluorescent probe in the detection of methyl orange.

[0019] Furthermore, detection is achieved based on the internal filtration effect: the fluorescence emission spectrum of the bistable copper nanoclusters overlaps with the absorption spectrum of methyl orange; methyl orange selectively quenches the fluorescence intensity of the bistable copper nanoclusters at 638 nm, while the fluorescence intensity of the carbon dots at 448 nm remains stable; the fluorescence intensity ratio F is monitored. 638 / F 448 The changes enable quantitative detection of methyl orange.

[0020] Furthermore, during detection, the sample to be tested is mixed with the dual-colorimetric fluorescent probe, and its fluorescence emission spectrum in the range of 350nm to 690nm is measured at an excitation wavelength of 345nm. The ratio of fluorescence intensity at 638nm to 448nm is calculated, and the concentration of methyl orange in the sample is determined according to the established ratio-concentration standard curve.

[0021] Furthermore, the application also includes visual semi-quantitative detection: by observing the color change of the dual-colorimetric fluorescent probe after it interacts with different concentrations of MO under ultraviolet light or sunlight, and comparing it with a standard color card, rapid on-site screening of methyl orange can be achieved.

[0022] Furthermore, the sample to be tested is an environmental water sample, including tap water, river water, or industrial wastewater.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: The CDs-Cu NCs fluorescent probe provided by this invention is synthesized from bistable copper nanoclusters and carbon dots through an in-situ synthesis method. The bistable copper nanoclusters utilize docosapentaenoic acid (DPA) and BSA as dual stabilizers, resulting in copper nanoclusters with higher stability. The prepared CDs-Cu NCs fluorescent probe exhibits superior detection performance and high sensitivity. Under optimal conditions, the detection limit for methyl orange is 0.06 μM, which is superior to traditional single-emission fluorescent probes, partial chromatography, and spectrophotometry, indicating higher sensitivity for detecting low-concentration pollutants. The probe shows a good linear relationship in its concentration response to methyl orange, covering a concentration range from nmol / L to μmol / L, making it suitable for quantitative analysis of water samples with varying degrees of pollution.

[0024] The CDs-Cu NCs fluorescent probe provided by this invention employs a dual emission ratio design, exhibiting strong anti-interference capability. This probe constructs a dual emission system based on the blue fluorescence of CDs at 448 nm and the orange-red fluorescence of DPA-Cu NCs at 638 nm. The fluorescence emission spectrum of DPA-Cu NCs overlaps with the absorption spectrum of MO. MO selectively quenches the fluorescence intensity of DPA-Cu NCs at 638 nm, while the fluorescence intensity of CDs at 448 nm remains stable. The fluorescence intensity ratio F is utilized... 638 / F 448 As a detection signal, it effectively eliminates interference caused by factors such as light source fluctuations, instrument drift, and probe concentration changes, thus improving the accuracy and repeatability of detection. In the presence of various substances including metal ions, dyes, sugars, and amino acids, the probe's response signal to methyl orange shows no significant change, indicating its excellent specific recognition ability and its ability to effectively eliminate interference in complex matrices.

[0025] The CDs-Cu NCs fluorescent probe provided by this invention exhibits strong stability and good environmental adaptability. Within a pH range of 3–11, the intensity ratio of the probe's two emission peaks remains essentially unchanged, making it suitable for actual water samples with varying pH levels. When stored under light-protected conditions for several days or subjected to multiple measurements within a short period, the fluorescence signal shows no significant attenuation, indicating good photochemical stability and suitability for batch detection and long-term storage. Within a NaCl concentration range of 0–1 mol / L, the probe signal shows no significant fluctuations, demonstrating strong resistance to ionic interference and suitability for saline or complex aquatic environments.

[0026] The CDs-Cu NCs fluorescent probe provided by this invention has strong visualization detection capabilities and is convenient for field application. Under 302 nm or 365 nm ultraviolet light irradiation, the fluorescence color on the probe solution or filter paper gradually changes from blue-violet to blue as the concentration of methyl orange increases, eventually tending towards colorless; under sunlight, the color of the filter paper transitions from colorless to orange. The color gradient change is obvious and suitable for visual interpretation; combined with RGB analysis, the color change of the filter paper has a good linear relationship with the MO concentration, and semi-quantitative or quantitative detection can be achieved by comparing with a standard color card, making it suitable for rapid on-site screening and compensating for the lack of portability of large instruments. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The figures show the spectral analysis of fluorescent nanoprobes; in the figure, A is the FT-IR spectrum of CDs-Cu NCs, CD, DPA-Cu NCs and DPA; B is the UV-Vis absorption spectrum of CD, DPA-Cu NCs and CDs-Cu NCs; C is the UV-Vis absorption spectrum of CD, DPA-Cu NCs+CD+MO and MO.

[0029] Figure 2 The structure of the fluorescent nanoprobes is characterized; in the figure, A is the transmission electron microscope image of CDs-Cu NCs, B is the transmission electron microscope image of DPA-Cu NCs, C is the transmission electron microscope image of DC, D is the particle size distribution of CDs-Cu NCs, E is the particle size distribution of DPA-CuNCs, and F is the particle size distribution of DC.

[0030] Figure 3 The fluorescence spectra of CDs-Cu NCs, CD, and DPA-Cu NCs are shown; the insets are fluorescence color photographs under a 360 nm UV lamp, where A is the fluorescence color of CDs-Cu NCs, B is the fluorescence color of CD, and C is the fluorescence color of DPA-Cu NCs.

[0031] Figure 4 The fluorescence signals of CDs-Cu NCs under different excitation wavelengths are shown.

[0032] Figure 5The results show the comparison between high concentration and blank experiments; in the figure, A is the comparison between high concentration and blank experiments of CD; B is the comparison between high concentration and blank experiments of copper clusters; and C is the comparison between high concentration and blank experiments of CDs-Cu NCs.

[0033] Figure 6 The results show the probe's response to the MO concentration gradient; in the figure, A is the response of the fluorescence emission spectrum of CDs-CuNCs to the MO concentration change; B is the response of the fluorescence emission spectrum of CD to the MO concentration change; C is the linear fit of the effect of the MO concentration gradient on CDs-CuNCs; D is the linear fit of the effect of the MO concentration gradient on CD; E is the bimodal CIE plot; and F is the unimodal CIE plot.

[0034] Figure 7 The figures show the fluorescence spectrum of the mixture of DPA-Cu NCs and CD and the fluorescence excitation spectrum in response to MO; in the figures, A is the fluorescence spectrum of the mixture of DPA-Cu NCs and CD at a volume ratio of 2:1 and the fluorescence excitation spectrum in response to MO; B is the fluorescence spectrum of the mixture of DPA-Cu NCs and CD at a volume ratio of 1:2 and the fluorescence excitation spectrum in response to MO.

[0035] Figure 8 The figures show the excitation fluorescence spectra of CDs-Cu NCs solution before and after centrifugation and dialysis. In the figure, A is the excitation fluorescence spectrum of CDs-Cu NCs solution before centrifugation; B is the excitation fluorescence spectrum of CDs-Cu NCs solution after centrifugation; C is the excitation fluorescence spectrum of CDs-Cu NCs solution before dialysis; and D is the excitation fluorescence spectrum of CDs-Cu NCs solution after dialysis. The inset in the figure is a fluorescence color photograph under a 360nm ultraviolet lamp.

[0036] Figure 9 The figures show the stability and anti-interference performance of the probe. In the figure, A is a line graph showing the effect of pH on the excitation fluorescence of CDs-Cu NCs; B is a line graph showing the effect of time on the excitation fluorescence of CDs-Cu NCs for 2 hours; C is a line graph showing the effect of time on the excitation fluorescence of CDs-Cu NCs for 2 days; and D is a bar graph showing the effect of salt solution on the excitation fluorescence of CDs-Cu NCs.

[0037] Figure 10 The figure shows the effect of potential interfering substances on the MO sensing system; in the figure, A is a fluorescence intensity bar chart of the effect of 120 μM potential interfering substance on the 60 μM MO sensing system; B is a fluorescence intensity bar chart of the interference of 120 μM potential interfering substance on the 60 μM MO sensing system.

[0038] Figure 11The results show the sensing mechanism analysis. In the figure, A is the UV absorption spectrum of MO and the fluorescence emission spectrum of CDs-Cu NCs; B is the UV-Vis absorption spectrum of CDs-Cu NCs, CDs and CDs-Cu NCs+MO.

[0039] Figure 12 The results are shown in the dual-color fluorescence control experiment. In the figure, A shows the corresponding fluorescence reaction color change under sunlight and the fluorescence change spectrum of CD plus MO under a 365nm UV lamp; B shows the corresponding fluorescence reaction color change under sunlight and the fluorescence change spectrum of DPA-CuNCs plus MO under a 365nm UV lamp; C shows the linear correlation graph of the fluorescence sensing plateau between different concentrations of MO and CD; D shows the linear correlation graph of the fluorescence sensing plateau between different concentrations of MO and DPA-CuNCs. Detailed Implementation

[0040] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0041] Example 1: A dual-colorimetric fluorescent probe for detecting methyl orange, its preparation method and application.

[0042] I. Test Reagents and Instruments D-Penicillamine was purchased from Shanghai Titan Technology Co., Ltd.; copper nitrate trihydrate CuSO4·3H2O was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; bovine serum albumin (BSA) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0043] The following instruments were purchased: DF-101S thermostatic magnetic stirrer (purchased from Gongyi Yuhua Instrument Co., Ltd.); electric drying oven (purchased from Shanghai Yiheng Scientific Instrument Co., Ltd.); H1850 high-speed benchtop centrifuge (purchased from Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); FD-1-50 vacuum freeze dryer (purchased from Beijing Boyikang Experimental Instrument Co., Ltd.); UV-9000S ultraviolet-visible spectrophotometer (purchased from Shanghai Yuanxi Instrument Co., Ltd.); F-4700 fluorescence spectrophotometer (purchased from Hitachi High Technology Scientific Knock Plant, Japan); WFH-203D projection reflectance analyzer (purchased from Shanghai Chitang Industrial Co., Ltd.); and WQF-530A Fourier transform infrared spectrometer (purchased from Beijing Beifen Ruili Analytical Instrument Co., Ltd.).

[0044] II. Test Methods 1. Preparation and Synthesis of Fluorescent Nanomaterials 1.1 Preparation of Bistable Copper Nanoclusters Weigh 0.02388 g of docosapentaenoic acid (DPA) into a beaker, add 16.0 mL of deionized water to dissolve it, and the concentration is 0.01 mol / L. Under vigorous stirring, add 160 μL of 0.1 mol / L Cu(NO3)2 solution dropwise. Continue stirring the mixture for 80 min. Then, add 0.4 mL of 0.8 mg / mL BSA solution dropwise while stirring. After stirring for 10 min, place the mixture in deionized water under light-protected conditions for dialysis for 24 h. The molecular weight cutoff of the dialysis bag is 8 kDa to remove unreacted substances and small molecule byproducts, and obtain DS-CuNCs solution.

[0045] 1.2 Synthesis of Carbon Dot CD Bermuda grass collected from pastures and park lawns was dried, pulverized, and ground, then passed through an 80-mesh sieve to obtain grass powder. 2g of grass powder was dispersed in 40mL of deionized water and centrifuged at 5000rpm for 15min to obtain a homogeneous suspension. The resulting suspension was transferred to a 100mL stainless steel autoclave lined with polytetrafluoroethylene (PTFE) and heated at 180℃ for 8h, then naturally cooled to 25℃. The reaction mixture was collected in 100mL centrifuge tubes and centrifuged at 10000rpm for 5min to remove insoluble impurities. The supernatant was filtered sequentially through 0.45μm and 0.22μm filter membranes to further remove residual particles, yielding a filtrate. To remove small molecule byproducts and inorganic salts, the filtrate was placed in a dialysis bag with a molecular weight cutoff of 8kDa and dialyzed with deionized water for 24h, with the water changed every 6h, to obtain a CD solution. The CD solution was divided into three equal parts and freeze-dried at -40℃ for 12h to obtain powdered CD for subsequent structural and optical characterization.

[0046] 1.3 In-situ synthesis of carbon dot / copper nanocluster composite probes CDs-Cu NCs Add 10 mL of the prepared CD solution to DS-Cu NCs, stir for 10 min, and then dialyze in deionized water under light-protected conditions for 24 h. The molecular weight cutoff of the dialysis bag is 8 kDa to remove unreacted substances and small molecule byproducts, and obtain CDs-Cu NCs solution, which is stored at 4 °C for later use.

[0047] 2. Characterization of fluorescent nanomaterials 2.1 Morphology and Particle Size Analysis Take 10 mL of each of the CD, CDs-Cu NCs, and DPA-Cu NCs solutions and place them in 50 mL centrifuge tubes. Freeze the tubes for later use. Seal the three frozen solutions with plastic wrap, poke holes in the wrap, and freeze-dry them at -40°C for 48 hours. Send the freeze-dried solutions for analysis to obtain transmission electron microscopy (TEM) images. Mark the images in particle size distribution calculation software and export the data. Plot the particle size distribution in Origin.

[0048] 2.2 Infrared and Ultraviolet Spectroscopy Detection Take three 5 mL aliquots of DPA-Cu NCs, CD and CDs-Cu NCs solutions respectively. Preheat the UV-Vis spectrometer for 15 min, calibrate with deionized water, and record and save the data.

[0049] Take 50 mL of DPA-Cu NCs, CD, and CDs-Cu NCs solutions respectively and place them in centrifuge tubes with sealed membrane inserts. Freeze at -20°C for 24 hours. After freezing, place the solutions on a shelf and check the seal. Run the freeze dryer for 30 minutes, then turn on the vacuum pump, disconnect the vent tube, and freeze-dry the solutions at -40°C for 2 days. Seal and store the freeze-dried DPA-Cu NCs, CD, and CDs-Cu NCs powders for later use. Weigh three 70 mg portions of potassium bromide, dry at 60°C for 4 hours, and compress into tablets for later use. Weigh 3 mg of each of the DPA-Cu NCs, CD, and CDs-Cu NCs powders and analyze them using an infrared spectrometer. Save the data for later use.

[0050] 3. Construction of ratiometric fluorescent probes and optimization of detection conditions 3.1 Determination of the optimal excitation wavelength Take 200 μL of CDs-Cu NCs solution and place it in a fluorescence spectrophotometer. Set the excitation wavelength to ex=365 nm, the scanning range to λ=ex+10-2ex-10, and the slit width to 5.0 nm. Then, detect the excitation wavelengths at ex=335 nm, 345 nm, 355 nm, 375 nm, and 385 nm. The optimal excitation spectrum is obtained by comparison.

[0051] 3.2 High-concentration control test MO was detected using three probes: CD, DPA-Cu NCs, and CDs-Cu NCs. The CD solution was diluted 13-fold, the CDs-CuNCs solution 2-fold, and the DPA-Cu NCs solution 2-fold for subsequent experiments to reduce errors in later testing. 400 μL of each of the three solutions were used as blanks; 400 μL of each solution were also mixed with 25 μL of MO to represent high-concentration solutions. The blank and high-concentration solutions were measured using a fluorescence spectrophotometer, with optimal excitation at ex=345 nm and a slit width of 5.0 nm. Data were retained for later use.

[0052] 3.3 Probe response to MO concentration gradient To demonstrate the responsiveness of CDs-Cu NCs and CDs to MO, i.e., the fluorescence emission spectrum, a 1 mmol / L MO solution was prepared and diluted to concentrations of 1 mmol / L, 800 μmol / L, 600 μmol / L, 400 μmol / L, 200 μmol / L, 100 μmol / L, 80 μmol / L, 60 μmol / L, 40 μmol / L, 20 μmol / L, 10 μmol / L, 8 μmol / L, 6 μmol / L, and 4 μmol / L, respectively. Dissolved in 1 mol / L, 2 μmol / L, 1 μmol / L, 800 nmol / L, 600 nmol / L, 400 nmol / L, 200 nmol / L, 100 nmol / L, 80 nmol / L, 60 nmol / L, 40 nmol / L, 20 nmol / L, 10 nmol / L, 8 nmol / L, 6 nmol / L, 4 nmol / L, 2 nmol / L, and 0 nmol / L solutions, respectively, and then incubated at room temperature for 3 min. The fluorescence intensity of the solutions in the range of 350 nm to 690 nm was recorded using a fluorescence spectrophotometer with an excitation wavelength of 345 nm and a slit width of 5.0 nm. The detection data were then saved.

[0053] 3.4 Determination of the optimal ratio of DPA-Cu NCs and CD To assess the stability of the methyl orange response after mixing copper clusters and carbon dots at different concentrations, 2 mL of DPA-CuNCs solution and 1 mL of CD solution were mixed to obtain a 2:1 concentration ratio mixture. This mixture was then further mixed with methyl orange solutions of different concentration gradients and incubated at room temperature for 3 min. The fluorescence intensity of the solution in the range of 350 nm to 690 nm was recorded using a fluorescence spectrophotometer with an excitation wavelength of 345 nm and a slit width of 5.0 nm. The detection data were then saved.

[0054] Take 1 mL of DPA-Cu NCs solution and 2 mL of CD solution, mix the two solutions to obtain a mixed solution with a concentration ratio of 1:2, and test it in the same way as above.

[0055] 4. Stability and anti-interference performance test of the probe 4.1 The effect of centrifugation dialysis on probe performance To investigate the effect of centrifugation and dialysis separation of precipitates and impurities on the solution, a 10 cm section of an 8 kDa dialysis bag was cut and placed in a 100 mL beaker. 80 mL of deionized water was added, and the beaker was placed in a constant-temperature magnetic stirrer at 35°C for 5 min. After sufficient softening and dialysis, 10 mL of CDs-Cu NCs solution was placed in the dialysis bag, sealed with a dialysis clamp, and a certain amount of deionized water was added to the beaker. The mixture was allowed to stand in the dark for 24 h, with the water changed every 6 h. Another 10 mL of the CDs-Cu NCs solution was placed in a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 5 min to remove insoluble impurities. The fluorescence intensity of the centrifuged and dialyzed CDs-Cu NCs solutions in the range of 350 nm to 690 nm was recorded by a fluorescence spectrophotometer, with the original CDs-Cu NCs solution as a control. The excitation wavelength was 345 nm and the slit width was 5.0 nm. The detection data were saved for subsequent control analysis.

[0056] 4.2 Environmental Factor Impact Test To investigate the effect of pH on CDs-Cu NCs, 10 mL of 0.1 mol / L sodium hydroxide solution and 10 mL of 0.1 mol / L dilute sulfuric acid solution were prepared and placed in beakers for later use. First, the initial pH value of the CDs-Cu NCs was measured. Then, four 5 mL aliquots of the CDs-Cu NCs solution were taken, and the pH of the original solution was titrated to 3, 5, 9, and 11 respectively using the prepared sodium hydroxide and dilute sulfuric acid solutions. The fluorescence intensity of the solutions in the range of 350 nm to 690 nm was recorded using a fluorescence spectrophotometer with an excitation wavelength of 345 nm and a slit width of 5.0 nm. The detection data were saved for subsequent comparative analysis.

[0057] To investigate the effect of time on CDs-Cu NCs, two 20 mL aliquots of in-situ synthesized CDs-Cu NCs solutions were taken and stored in the dark. One aliquot was measured every 2 minutes for a 2-hour period, and the other was measured every 2 days for a 2-day period. Fluorescence intensity in the solution was recorded using a fluorescence spectrophotometer in the range of 350 nm to 690 nm, with an excitation wavelength of 345 nm and a slit width of 5.0 nm. The data were saved for subsequent comparative analysis.

[0058] To investigate the effect of salt solutions on CDs-Cu NCs, sodium chloride was selected as the experimental reference. 5.85 g of sodium chloride was dissolved in 100 mL of deionized water to prepare a 1 mmol / L sodium chloride solution, which was then successively diluted to 0.8 mmol / L, 0.6 mmol / L, 0.4 mmol / L, and 0.2 mmol / L. 200 μL of each prepared sodium chloride solution was mixed with 200 μL of CDs-Cu NCs solution, respectively. 200 μL of each prepared sodium chloride solution was also mixed with 200 μL of deionized water as blanks. The fluorescence intensity of the solution in the range of 350 nm to 690 nm was recorded using a fluorescence spectrophotometer with an excitation wavelength of 345 nm and a slit width of 5.0 nm. The detection data were saved for subsequent comparative analysis.

[0059] 4.3 Selectivity and Interference Tests To verify the selectivity of the established method for MO, dimethyl orange, fructose, bromocresol green, and Cu were prepared at a concentration of 120 μM. 2+ Cr 3+ Methyl red, Sudan III, Ni 2+ Glutamic acid, Mg 2+ Sudan IV, tetracycline, Pb 2+ Fe 3+ Sudan I, Ca 2+ Sudan II, Hg 2+ sucrose, glucose and Co 2+ The above solutions were used to replace MO in the reaction with CDs-Cu NCs. 200 μL of the solutions of the various substances mentioned above were mixed with 200 μL of CDs-Cu NCs solution diluted 2 times, and allowed to stand for 2 min. After the reaction was completed, the fluorescence intensity was measured by a fluorescence spectrophotometer with an excitation wavelength of 345 nm and a slit width of 5.0 nm.

[0060] To evaluate the anti-interference capability of the detection system, dimethyl orange, fructose, bromocresol green, and Cu were added to MO-containing CDs-Cu NCs. 2+ Cr 3+ Methyl red, Sudan III, Ni 2+ Glutamic acid, Mg 2+Sudan IV, tetracycline, Pb 2+ Fe 3+ Sudan I, Ca 2+ Sudan II, Hg 2+ sucrose, glucose and Co 2+ The solution, with a concentration of 120 μM, was tested using the same method as above.

[0061] 5. Research on sensing mechanisms To verify the experimental theory, 1 mL of CDs-Cu NCs solution and 1 mL of 0.1 mol / L MO solution were taken and allowed to react at room temperature for 3 min. The fluorescence intensity was measured using a fluorescence spectrophotometer with an excitation wavelength of 345 nm and a slit width of 5.0 nm. The detection data were saved for subsequent control analysis. MO, CDs-Cu NCs, and DPA-Cu NCs solutions of the same concentration and volume were also measured using a UV-Vis spectrophotometer from 200 nm to 700 nm, and the data were saved.

[0062] 6. Actual sample testing and visualization applications 6.1 Real water sample spike recovery test Prepare pulsating agents, 75% ethanol, 10% glucose solution, and tap water as real interfering substances. Dilute the pulsating agents, 75% ethanol, 10% glucose solution, and tap water 1000 times with deionized water for later use. Dissolve 3.27g of MO in 100mL of deionized water to prepare a 0.1mol / L MO solution. Dilute the solution fresh before use. Take eight 5mL portions of CDs-Cu NCs solution, add 5mL of 200μm / L MO solution, let stand for 3min, then add a certain amount of real interfering substances to each, let stand for 3min, and measure the fluorescence intensity using a fluorescence spectrophotometer with an excitation wavelength of 345nm and a slit width of 5.0nm for subsequent control analysis.

[0063] 6.2 Visual Detection Based on Fluorescence Color Changes Take CDs-Cu NCs and CD solutions, and add MO solutions of different concentrations to change the fluorescence color of the CDs-Cu NCs solution from blue-violet to red, and the fluorescence color of CD from blue to colorless. Add the prepared solutions dropwise onto square filter paper cut to 1cm x 1cm with equal width and height, place them in a vacuum drying oven, dry at 40℃ for 2 hours, and then place them under sunlight. Arrange them in order of solution concentration from low to high, and photograph them for later use. Then place the filter paper in a transmission reflectance analyzer, emitting wavelengths of 254nm and 365nm, and photograph them for later use.

[0064] III. Test Results 1. Synthesis and structural characterization of fluorescent nanoprobes Figure 1a represents the FT-IR spectra of CDs-Cu NCs, CD, DPA-Cu NCs, and DPA; b represents the FT-IR spectra of DPA-Cu NCs, CD, and CDs-Cu NCs; and c represents the UV-Vis absorption spectra of CD, CDs-Cu NCs, and MO.

[0065] The infrared spectrum of DPA shows that the characteristic absorption peak of thiol-SH is 2575±25 cm⁻¹. -1 The stretching vibration peak of amino-NH2 is 3650±350 cm⁻¹. -1 In pure BSA, the peak position of amide I is at 1650 cm⁻¹. -1 The peak position of amide II band is at 1540 cm⁻¹. - 1. After synthesizing DPA-Cu NCs, the -SH peak weakened or even disappeared, and the peak positions of -NH2 and amide I and II bands also shifted, indicating that DPA, BSA, and Cu... 2+ Successful combination, DPA-Cu NCs were successfully synthesized.

[0066] The infrared spectrum of CD showed a value of 3300±100 cm⁻¹. -1 The broad peak, and 1730±10cm -1 The peak indicates that CD has many hydrophilic groups on its surface, which can stably disperse in water; the electronic effect of these groups is an important reason why CD can emit blue fluorescence, indicating that the synthesized CD meets the experimental requirements.

[0067] In the infrared spectrum of CDs-Cu NCs, the characteristic peaks of CD and DPA-Cu NCs appear simultaneously, indicating that the two materials can coexist stably in the composite system without destructive interactions, laying the structural foundation for the subsequent realization of the "dual-emission probe" function. Furthermore, in the ultraviolet spectrum, MO has a strong characteristic absorption peak at 470±10 nm, while the fluorescence emission peak of DPA-Cu NCs is at 638 nm, and that of CD is at 448 nm. The peaks of MO do not overlap with those of DPA-Cu NCs and CD, further confirming the stability and accuracy of the experiment.

[0068] In the UV-Vis absorption spectra, the linear conditions of CD and DPA-Cu NCs, CD and CDs-Cu NCs are highly coincident and show a unified trend in the characteristic peaks. Moreover, the characteristic peaks overlap, which once again confirms the stability and accuracy of our experiment.

[0069] The morphology and size of DPA-Cu NCs, CD, and CDs-Cu NCs solutions were analyzed using transmission electron microscopy (TEM) and particle size distribution analysis. Figure 2The C and F values ​​show that CD particles have regular morphology and a concentrated and narrow particle size distribution, indicating that CD has good size uniformity during synthesis and no obvious agglomeration. Figure 2 B and E results show that the particle size of DPA-Cu NCs is slightly smaller than that of CD, and the particles are uniformly dispersed. This is because BSA modification effectively inhibits the aggregation of nanoclusters, laying the foundation for subsequent mixing with CD. Figure 2 The A and D results show that the CDs-Cu NCs particles did not show obvious aggregation, and the particle size distribution also conforms to the characteristics of the "CD+DPA-Cu NCs" composite system, proving that CD and DPA-Cu NCs can coexist stably in solution without mutual precipitation or agglomeration, thus ensuring the stability of the probe system.

[0070] Figure 3 Fluorescence spectroscopy revealed that DPA-Cu NCs exhibited a maximum emission wavelength at 638 nm. Inset c showed that it emitted orange-red fluorescence under 302 nm UV irradiation, which is related to the modification effect of DPA on its surface and the nanocluster structure. CDs showed a maximum emission peak at 448 nm. Inset b showed that it emitted bright blue fluorescence under 302 nm UV irradiation, attributed to the size effect of carbon dots and surface functionalized groups. When the two were mixed in situ to form a CDs-Cu NCs ratiometric fluorescent probe, dual emission peaks at 448 nm and 638 nm appeared. Inset a showed that it emitted purple-red fluorescence under 302 nm UV irradiation, proving that the dual fluorescent units were successfully combined and the probe system was completed.

[0071] 2. Optimization of the detection performance of the ratiometric fluorescent probe and establishment of calibration curves 3.1 Determination of the optimal excitation wavelength By testing the probe fluorescence signals at different excitation wavelengths (335 nm, 345 nm, 355 nm, 365 nm, 375 nm, and 385 nm), it was found that at 345 nm excitation, the fluorescence intensity of the dual emission peaks at 448 nm and 638 nm both reached their maximum values, with clear peak shapes and no interference from extraneous peaks. The results are shown in [Figure number missing]. Figure 4 Therefore, 345 nm was determined as the optimal excitation wavelength to provide uniform conditions for all subsequent detection experiments and reduce errors caused by differences in excitation wavelength.

[0072] 3.2 Blank and High-Concentration Control Experiment The fluorescence properties of the material and the stability of the system were verified by comparing the fluorescence emission spectra of the high-concentration state and the blank state. The results are shown in [Figure number missing]. Figure 5 .

[0073] Figure 5Figure A shows a significant difference in signal between the high concentration CD and the blank state, indicating good fluorescence stability. In the high concentration CD group, after diluting CD 13 times and mixing it with MO, its fluorescence intensity was much lower than that of the blank group. In the blank group, after diluting CD 13 times, a high-intensity characteristic emission peak appeared at 448 nm, with a symmetrical peak shape and no impurities, indicating that CD has a strong response to MO.

[0074] Figure 5 B shows that after diluting DPA-Cu NCs by 2 times and exciting them with 345 nm, a strong fluorescence emission peak appears at 638 nm. The peak shape is also round, symmetrical and without impurity peaks. However, compared with the blank experiment, there is no significant difference between the two, which indicates that the copper cluster is very stable.

[0075] Figure 5 The C-values ​​show that CDs-Cu NCs can maintain the core characteristics of dual emission peaks at high concentrations. The fluorescence signals of CD and DPA-Cu NCs are independent and stable without mutual interference, proving that the synergistic effect of the "dual signal units" of the ratiometric fluorescent probe is effective. This lays the foundation for the subsequent detection of MO by the "dual peak intensity ratio" and can also ensure the reliability of subsequent MO concentration gradient response experiments.

[0076] 3.3 Probe response to MO concentration gradient Figure 6 As shown in Figure A, the fluorescence intensity of CDs-Cu NCs at 448 nm decreased continuously with increasing MO concentration, while the fluorescence intensity at 638 nm remained essentially unchanged. Figure 6 The changes in fluorescent spots can be observed more clearly in E and F. Figure 6 The D-display shows that the CD fluorescence intensity ratio F 638 / F 448 The correlation coefficient R shows a strong linear relationship with MO concentration. 2 A value greater than 0.991 indicates a good linear fit. Figure 6 The C-values ​​show that, through linear regression calculation, the detection limit of the CDs-Cu NCs probe is 0.06 μM, which is superior to the 0.01 μmol / L to 1 μmol / L of traditional single-emission fluorescent probes, the 0.1 mg / L to 1 mg / L of partial chromatography, and the 0.1 mg / L to 1 mg / L of spectrophotometry. It can accurately capture low concentrations of MO residues in water and meet the sensitivity requirements of actual environmental monitoring.

[0077] 3.4 Determination of the optimal ratio of DPA-Cu NCs and CD Figure 7 A shows that when the volume ratio of DPA-Cu NCs to CD is 2:1, the intensity of the dual emission peak signal is more balanced, and the ratio fluctuation caused by the change in MO concentration is more obvious. Figure 7Figure B shows that when the volume ratio of DPA-Cu NCs to CD is 1:2, the fluorescence intensity of CD is too high, masking the signal changes of DPA-Cu NCs and leading to a decrease in sensitivity. Comparison shows that a volume ratio of DPA-Cu NCs to CD of 2:1 is the optimal mixing ratio, ensuring that the probe maintains a stable reference signal during detection while allowing the response signal to fully reflect changes in MO concentration.

[0078] 4. Stability and anti-interference performance test of the probe Figure 8 As shown in Figures A through D, after centrifugation at 10,000 rpm for 5 min and dialyzing with a molecular weight cutoff of 1 kDa to 3.5 kDa for 24 h, the fluorescence spectrum of CDs-Cu NCs was basically consistent with that of the original solution, with no obvious peak shift or intensity decrease. This indicates that the removal of impurities and unreacted raw materials during the pretreatment process does not affect the probe performance and further ensures the accuracy of detection.

[0079] Figure 9 Figure A shows that the fluorescence intensity F of the double emission peak of CDs-Cu NCs varies with the solution pH from 3 to 11. 430 F 630 The ratios showed no significant changes, proving that the probes can work stably in both acidic and alkaline environments and are suitable for detecting actual water samples at different pH levels. Figure 9 As shown in B and C, the intensity and ratio of the dual emission peaks of CDs-Cu NCs remained basically constant over time without significant decay, indicating that the material has good photochemical stability and can be stored for a long time or used for batch testing. Figure 9 The D-values ​​show that the intensity of the dual emission peaks of CDs-Cu NCs does not fluctuate significantly within the NaCl concentration range of 0 mol / L to 1 mol / L, proving that they can still be stably detected in saline water, have strong resistance to ionic strength interference, and meet the needs of environmental monitoring.

[0080] Figure 10 Figure A shows that, under conditions where common interfering substances are present, including dyes such as methyl red, Sudan III, and bromocresol green, and metal ions such as Cu, the presence of these substances is significant. 2+ Fe 3+ Pb 2+ Hg 2+ The probe system selectively detects the MO signal, i.e., F, using carbohydrates—glucose and sucrose, and amino acids—glutamic acid. 630 / F 430 The ratio showed no significant fluctuation; similarly, in Figure 10 In B, under the influence of interfering substances, the probe system's detection signal for MO, i.e., F 630 / F 430The ratio showed no significant fluctuation. This indicates that CDs-Cu NCs exhibit strong selectivity and specificity for MO, effectively eliminating interference from coexisting substances and demonstrating outstanding selectivity.

[0081] 5. Research on sensing mechanisms from Figure 11 As shown in Figure A, the absorption peak of MO overlaps with the fluorescence emission peak of DPA-Cu NCs, which is precisely the core condition for the fluorescence internal filtration (IFE) effect. MO acts as a "light-absorbing substance," specifically absorbing the fluorescent photons emitted by DPA-Cu NCs, leading to a weakening of the fluorescence of DPA-Cu NCs, i.e., fluorescence quenching. Furthermore, fluorescence comparisons also show that the peak positions of DPA-Cu NCs and MO in the 400nm to 450nm range are essentially overlapping. Figure 11 The ultraviolet spectrum of B also confirmed the spectral overlap between the two, jointly proving that IFE is the core mechanism for the detection of MO by CDs-Cu NCs.

[0082] UV-Vis spectrophotometer measurements showed that the characteristic absorption spectrum of MO significantly overlapped with the fluorescence emission spectrum of DPA-Cu NCs at 638 nm, but not with the emission spectrum of CD at 448 nm. This provides a basis for the occurrence of the fluorescence internal filtering (IFE) effect. After adding MO to the probe system, the fluorescence intensity of DPA-Cu NCs at 638 nm decreased significantly with increasing MO concentration, while the fluorescence intensity of CD at 448 nm remained stable. Furthermore, the ratio of their fluorescence intensities, F1, was significantly higher. 638 / F 448 The signal also decreased systematically as MO concentration increased. This series of changes perfectly matches the mechanism of the internal filtration effect, indicating that the signal for detecting MO is dominated by IFE.

[0083] 6. Actual sample testing and visualization applications 6.1 Real water sample spike recovery test To verify the high accuracy and reliability of this experiment, simulated complex matrix samples such as pulsating liquid, alcohol, and sugar solution were used, and the spiked recovery method was employed. The test results are shown in Table 1. When the spike concentration was 50 μmol / L and 100 μmol / L, the recovery rate was between 97.04% and 100.54%, close to 100%, indicating that the probe detection results had little deviation from the actual MO content and no significant interference. The relative standard deviation (RSD) of precision was less than 2.955%, with some samples having an RSD of only 0.17%, demonstrating good repeatability and high data stability during batch detection, meeting the precise monitoring requirements of actual water samples.

[0084] Table 1. Measurement of MO in real samples

[0085] 6.2 Visual Detection Based on Fluorescence Color Changes Quantitative visual detection and rapid screening were achieved through a "dual-color fluorescence control" experiment: probe solutions with different MO concentrations were sequentially dropped onto filter paper and dried. Filter paper without MO appeared blue-purple under a projection-reflectance analyzer at emission wavelengths of 302nm and 365nm. This color is a mixture of the blue of CD and the orange-red of DPA-Cu NCs. As the MO concentration increased, the fluorescence of DPA-Cu NCs was quenched, and the color gradually turned blue. At high MO concentrations, the filter paper was nearly colorless. This color gradient change could be observed with the naked eye. Under sunlight, the color of the filter paper gradually transitioned from colorless to orange. Figure 12 A and B. RGB analysis results are shown in [link to results]. Figure 12 C, D, R 2 A value between 0.993 and 0.994 indicates that there is virtually no color difference when taking photos.

[0086] The above results show that the detection method provided by the present invention can achieve visualized quantitative analysis of MO by comparing with a standard color chart. It is suitable for rapid on-site screening and detection, making up for the lack of portability in instrument quantitative detection, and providing a practical technical tool for rapid and accurate monitoring of MO pollution in water bodies.

[0087] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments.

[0088] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A dual-colorimetric fluorescent probe for detecting methyl orange, characterized in that, The dual-colorimetric fluorescent probe is a carbon dot / copper nanocluster composite probe, which is synthesized from bistable copper nanoclusters and carbon dots through an in-situ synthesis method. The bistable copper nanoclusters are prepared by reacting docosapentaenoic acid and copper salt to form a precursor, which is then stabilized by bovine serum albumin, wherein docosapentaenoic acid and bovine serum albumin serve as dual stabilizers. The carbon dots are prepared from plant biomass by a hydrothermal method.

2. The dual-colorimetric fluorescent probe according to claim 1, characterized in that, The plant biomass is the root of Bermuda grass.

3. The method for preparing the dual-colorimetric fluorescent probe according to claim 1, characterized in that, Includes the following steps: Eicosapentaenoic acid solution was mixed with copper salt solution to form a precursor, which was then stabilized by adding bovine serum albumin solution and purified by dialysis to obtain bistable copper nanoclusters solution. Plant biomass is dried, pulverized, and sieved to obtain grass powder, which is then dispersed in water and centrifuged to obtain a uniform suspension. The suspension was subjected to a hydrothermal reaction, and the reaction product was purified by centrifugation, filtration, and dialysis to obtain a carbon dot solution. A carbon dot solution was mixed with a bistable copper nanocluster solution, and after stirring and reaction, the mixture was purified by dialysis to obtain a carbon dot / copper nanocluster composite probe.

4. The preparation method according to claim 3, characterized in that, The carbon dot solution and the bistable copper nanocluster solution are mixed in equal volume ratio.

5. The application of the dual-colorimetric fluorescent probe according to claim 1 in the detection of methyl orange.

6. The application according to claim 5, characterized in that, During detection, the sample to be tested is mixed with the dual-colorimetric fluorescent probe, and its fluorescence emission spectrum in the range of 350nm~690nm is measured at an excitation wavelength of 345nm. The ratio of fluorescence intensity at 638nm to 448nm is calculated, and the concentration of methyl orange in the sample is determined according to the established ratio-concentration standard curve.

7. The application according to claim 5, characterized in that, The application also includes visual semi-quantitative detection: by observing the color change of the dual-color fluorescent probe after it reacts with different concentrations of methyl orange under ultraviolet light or sunlight, and comparing it with a standard color card, rapid on-site screening of methyl orange can be achieved.

8. The application according to claim 5, characterized in that, The sample to be tested is an environmental water sample, including tap water, river water, or industrial wastewater.

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