Ratio-dependent up-conversion fluorescent nanoprobe for veterinary drug detection, and preparation method and application thereof
By designing a multi-layered ratiometric upconversion fluorescent nanoprobe, the reliability problem of veterinary drug residue detection was solved, and selective quantitative detection of furazolidone and malachite green was achieved. It has high biological tissue penetration depth and high signal-to-noise ratio, and is suitable for the detection of veterinary drug residues in water.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGDONG UNIV OF TECH
- Filing Date
- 2026-01-20
- Publication Date
- 2026-04-17
AI Technical Summary
The reliability of fluorescence detection of veterinary drug residues in existing technologies is low, and there is a lack of ratiometric upconversion fluorescent nanoprobes based on upconversion nanoparticles, resulting in insufficient reliability of detection results.
A ratiometric upconversion fluorescent nanoprobe was designed, comprising a core nanoparticle, an inner shell, a middle shell, and an outer shell from the inside out. The core nanoparticle has the chemical composition NaGdF4:Yb/Tm, the inner shell is NaYbF4, the middle shell is NaGdF4:Yb/Er, and the outer shell is NaYF4. Sodium alginate was modified by electrostatic adsorption. The preparation methods include coordination reaction and coprecipitation reaction.
It achieves selective detection of furazolidone and malachite green, has good quantitative ability, avoids light damage, has high biological tissue penetration depth and high signal-to-noise ratio, and is suitable for the detection of veterinary drug residues in water.
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Figure CN121877831A_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the field of fluorescence detection technology, and particularly relates to ratiometric upconversion fluorescent nanoprobes for veterinary drug detection, their preparation methods, and applications. Background Technology
[0002] Antibiotics and other veterinary drugs play a crucial role in preventing the spread of harmful microorganisms and treating various diseases. They are used in the breeding of livestock, aquatic animals, and poultry such as pigs, cattle, sheep, fish, shrimp, chickens, and ducks. However, some veterinary drugs are not fully utilized by livestock, aquatic animals, or poultry, resulting in their release into the environment. Some veterinary drugs may also cause water and food chain pollution, and further accumulate in the human body through contaminated food, interfering with normal metabolism, destroying the microbial ecosystem, and directly endangering human health. Therefore, it is necessary to test the residual levels of veterinary drugs in water.
[0003] Fluorescence detection is an ideal method for detecting veterinary drug residues, enabling the detection of target analytes. Unlike traditional fluorescent probes, upconversion nanoparticles exhibit anti-Stokes luminescence properties, absorbing long-wavelength, low-energy light and emitting short-wavelength, high-energy light, effectively avoiding interference from background fluorescence and significantly improving fluorescence detection sensitivity. Furthermore, upconversion nanoparticles offer advantages such as tunable luminescence properties, high photochemical stability, low biotoxicity, narrow emission peaks, and long fluorescence lifetimes. Therefore, upconversion fluorescent nanoprobes based on upconversion nanoparticles have promising potential applications in the field of veterinary drug residue detection.
[0004] In ratiometric fluorescent probes, during fluorescence detection, one fluorescence signal intensity changes with the concentration of the target analyte, while the other fluorescence signal intensity remains constant. Quantitative detection of the target analyte can be achieved by measuring the ratio of the fluorescence signal intensities at the two wavelengths. Ratiometric fluorescent probes, through a self-calibration mechanism, effectively counteract the influence of non-specific factors such as environmental and instrument fluctuations on the fluorescence detection results, making them a more reliable type of fluorescent probe. However, currently, there is a lack of ratiometric upconversion fluorescent nanoprobes based on upconversion nanoparticles for the fluorescence detection of veterinary drug residues, resulting in the relatively low reliability of current fluorescence detection results for veterinary drug residues. Summary of the Invention
[0005] In view of this, this application provides a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection, its preparation method, and its application, in order to solve the technical problem of low reliability of fluorescence detection of veterinary drug residues in the prior art.
[0006] The first aspect of this application provides a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection, comprising, from the inside out: a core nanoparticle, an inner shell, a middle shell, and an outer shell;
[0007] The inner shell encapsulates the core nanoparticles, the middle shell encapsulates the inner shell, and the outer shell encapsulates the middle shell.
[0008] The chemical composition of the nuclear nanoparticles is: NaGdF4:Yb / Tm;
[0009] The chemical composition of the inner shell is: NaYbF4;
[0010] The chemical composition of the intermediate shell is: NaGdF4:Yb / Er;
[0011] The chemical composition of the outer shell is NaYF4.
[0012] Preferably, the molar ratio of Gd:Yb:Tm in the nuclear nanoparticles is 47~69.5:30~50:0.5~3;
[0013] In the intermediate shell, the molar ratio of Gd:Yb:Er is 47~69.5:30~50:0.5~3.
[0014] Preferably, the particle size of the nuclear nanoparticles is 10~30 nm;
[0015] The thickness of the inner shell is 3~10 nm;
[0016] The thickness of the intermediate shell is 1~10 nm;
[0017] The thickness of the outer shell is 3~10 nm.
[0018] Preferably, the outer shell of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection is modified with sodium alginate.
[0019] Preferably, the outer shell of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection is modified with sodium alginate through electrostatic adsorption.
[0020] Preferably, the ratiometric upconversion fluorescent nanoprobe used for veterinary drug detection has a particle size of 17-80 nm.
[0021] The second aspect of this application provides a method for preparing a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection, which can prepare the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection described in the first aspect, comprising the following steps:
[0022] Preparation steps of the nuclear rare earth source precursor solution: dissolve gadolinium salt, ytterbium salt and thulium salt in oleic acid and 1-octadecene to carry out coordination reaction to obtain the nuclear rare earth source precursor solution;
[0023] Preparation steps of the inner shell rare earth source precursor solution: Dissolve ytterbium salt in oleic acid and 1-octadecene to carry out a coordination reaction to obtain the inner shell rare earth source precursor solution.
[0024] Preparation steps of the intermediate shell rare earth source precursor solution: dissolve gadolinium salt, ytterbium salt and erbium salt in oleic acid and 1-octadecene to carry out coordination reaction to obtain the intermediate shell rare earth source precursor solution.
[0025] Preparation steps of the outer shell rare earth source precursor solution: Dissolve yttrium salt in oleic acid and 1-octadecene to carry out a coordination reaction to obtain the outer shell rare earth source precursor solution;
[0026] Preparation steps of nuclear nanoparticles: Alkaline fluorine source and alkaline sodium source are added to the nuclear rare earth source precursor solution for co-precipitation reaction to obtain nuclear nanoparticles;
[0027] The inner shell coating process involves adding an alkaline fluorine source, an alkaline sodium source, and core nanoparticles to the rare earth source precursor solution of the inner shell for co-precipitation reaction to obtain core nanoparticles coated with the inner shell.
[0028] The intermediate shell coating process involves adding an alkaline fluorine source, an alkaline sodium source, and the core nanoparticles coating the inner shell to a rare earth source precursor solution for the intermediate shell for co-precipitation reaction, thereby obtaining core nanoparticles that sequentially coat the inner shell and the intermediate shell.
[0029] The outer shell coating process involves adding an alkaline fluorine source, an alkaline sodium source, and core nanoparticles that coat the inner and middle shells to the rare earth source precursor solution of the outer shell for co-precipitation reaction. This yields core nanoparticles that sequentially coat the inner, middle, and outer shells, which are the ratiometric upconversion fluorescent nanoprobes used for veterinary drug detection.
[0030] Preferably, in the method for preparing the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection, after the outer shell coating step, the method further includes the following steps:
[0031] Oleic acid ligand removal steps: The ratiometric upconversion fluorescent nanoprobe used for veterinary drug detection was added to a hydrochloric acid aqueous solution and subjected to sonication and centrifugation in sequence to obtain a ratiometric upconversion fluorescent nanoprobe used for veterinary drug detection with oleic acid ligand removed.
[0032] Modification steps for sodium alginate: Add the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection with oleic acid ligand removed to the sodium alginate solution and stir to obtain sodium alginate modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection.
[0033] Preferably, in the oleic acid ligand removal step, the concentration of the hydrochloric acid aqueous solution is 0.05~0.2 M, the ultrasonic temperature is 25~35℃, the time is 0.5~1.5 h, the centrifugation speed is 10000~20000 rpm, and the time is 20~40 min.
[0034] Preferably, in the sodium alginate modification step, the concentration of sodium alginate in the sodium alginate solution is 0.2~1 mg / mL, the solvent is an acetate-sodium acetate buffer solution with pH = 4.0~6.0, the stirring temperature is room temperature, and the stirring time is 10~20 min.
[0035] Preferably, in the preparation method of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection, the reaction temperature of the coordination reaction is 150~180℃ and the reaction time is 20~40 min.
[0036] Preferably, in the preparation method of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection, the coprecipitation reaction process includes: reacting sequentially at 40~60℃ for 20~40 min, at 100~120℃ for 15~40 min, and at 280~320℃ for 40~80 min under an argon atmosphere.
[0037] Preferably, in the preparation method of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection,
[0038] The alkaline fluorine source is selected from ammonium fluoride, and the alkaline sodium source is selected from sodium hydroxide;
[0039] Gadolinium salts are selected from at least one of acetate, nitrate, sulfate, and chloride; ytterbium salts are selected from at least one of acetate, nitrate, sulfate, and chloride; thulium salts are selected from at least one of acetate, nitrate, sulfate, and chloride; erbium salts are selected from at least one of acetate, nitrate, sulfate, and chloride; and yttrium salts are selected from at least one of acetate, nitrate, sulfate, and chloride.
[0040] The third aspect of this application provides the application of the ratiometric upconversion fluorescent nanoprobes for veterinary drug detection described in the first aspect in the detection of furazolidone or malachite green.
[0041] Preferably, the application specifically includes: the application of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection described in the first aspect in the detection of furazolidone or malachite green in an aqueous medium.
[0042] Preferably, the application specifically includes the following steps:
[0043] Step 1: Mix the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection described in the first aspect with furazolidone or malachite green standard solution of concentration gradient and perform upconversion fluorescence detection to establish a linear relationship between the upconversion emission intensity of furazolidone or malachite green standard solution of concentration gradient and fluorescent nanoprobe.
[0044] Step 2: Mix the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection described in the first aspect with the furazolidone or malachite green sample to be detected, and then perform upconversion fluorescence detection to obtain the upconversion emission intensity of the fluorescent nanoprobe in the furazolidone or malachite green sample to be detected.
[0045] Step 3: Substitute the upconversion emission intensity of the fluorescent nanoprobe in the furazolidone or malachite green sample to be tested into the linear relationship between the upconversion emission intensity of the furazolidone or malachite green standard solution and the fluorescent nanoprobe at the concentration gradient, and calculate the concentration of the veterinary drug in the furazolidone or malachite green sample to be tested.
[0046] Compared with existing technologies, this application provides a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection that includes at least the following beneficial effects:
[0047] 1. The ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in this application exhibits excellent selectivity after incubation with veterinary drugs such as furazolidone (FTD) and malachite green (MG), resulting in significant quenching of fluorescence intensity. It can be used for the detection of furazolidone and malachite green. As the concentration of furazolidone or malachite green increases, the fluorescence intensity of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection gradually decreases, enabling quantitative detection of furazolidone or malachite green. Furthermore, the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection can be excited by low-energy near-infrared light without causing photodamage, has a higher penetration depth into biological tissues, no background fluorescence interference, and a higher signal-to-noise ratio, making it a highly reliable fluorescent detection method for veterinary drug residues.
[0048] 2. The ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in this application is further modified with sodium alginate on its surface. Sodium alginate improves the dispersibility of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection in aqueous media, making it suitable for the detection of veterinary drug residues in water. Attached Figure Description
[0049] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0050] Figure 1 Infrared spectra of oleic acid-coated ratiometric upconversion fluorescent nanoprobes for veterinary drug detection, oleic acid-ligand-removed ratiometric upconversion fluorescent nanoprobes for veterinary drug detection, sodium alginate, and sodium alginate-modified ratiometric upconversion fluorescent nanoprobes for veterinary drug detection provided in Example 1 of this application.
[0051] Figure 2 Transmission electron microscopy images of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection with oleic acid ligand removed and the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection modified with sodium alginate provided in Example 1 of this application.
[0052] Figure 3 The upconversion fluorescence spectra of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection with oleic acid ligand removed, the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection modified with sodium alginate, and the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection modified with sodium alginate after incubation with furazolidone or malachite green provided in Example 1 of this application.
[0053] Figure 4 The ratiometric upconversion fluorescent nanoprobe for veterinary drug detection, modified with sodium alginate as provided in Example 1 of this application, is used for the detection of furazolidone, malachite green, florfenicol, thiamphenicol, tobramycin, streptomycin, erythromycin, amoxicillin, and furazolidone and malachite green, and its UV-Vis absorption spectrum after incubation.
[0054] Figure 5 The ratio-upconversion fluorescent nanoprobe modified with sodium alginate for veterinary drug detection provided in Example 1 of this application and its ratio-upconversion fluorescence response after incubation with furazolidone, florfenicol, thiamphenicol, tobramycin, streptomycin, erythromycin and amoxicillin;
[0055] Figure 6 The ratio-upconversion fluorescent nanoprobe modified with sodium alginate for veterinary drug detection provided in Example 1 of this application and its ratio-upconversion fluorescence response diagram after incubation with malachite green, florfenicol, thiamphenicol, tobramycin, streptomycin, erythromycin and amoxicillin;
[0056] Figure 7 The upconversion fluorescence spectrum of the sodium alginate-modified ratiomatic upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1 of this application after incubation with furazolidone at a concentration of 0-100 µM.
[0057] Figure 8 for Figure 7The upconversion fluorescence spectrum shown is a scatter plot of the ratio of fluorescence intensity at 361 nm to fluorescence intensity at 450 nm for furazolidone at concentrations of 0–100 µM.
[0058] Figure 9 To Figure 8 The linear response relationship is obtained by performing local linear fitting on the 0~20 μM interval of the scatter plot of the data shown.
[0059] Figure 10 To Figure 8 The linear response relationship is obtained by performing local linear fitting on the 30~80 μM interval of the scatter plot of the data shown.
[0060] Figure 11 The upconversion fluorescence spectrum of the sodium alginate-modified ratiomatic upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1 of this application after incubation with malachite green at a concentration of 0-150 µM.
[0061] Figure 12 for Figure 11 The upconversion fluorescence spectrum shown is a scatter plot of the ratio of fluorescence intensity at 540 nm to fluorescence intensity at 800 nm for malachite green with concentrations of 0–150 µM.
[0062] Figure 13 To Figure 12 The linear response relationship is obtained by performing local linear fitting on the 0~40 μM interval of the scatter plot of the data shown.
[0063] Figure 14 To Figure 12 The linear response relationship is obtained by performing local linear fitting on the 40~90 μM interval of the scatter plot of the data shown.
[0064] In the attached diagram, Figure 2 Figure (a) is a transmission electron microscope (TEM) image of a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection with oleic acid ligands removed, and Figure (b) is a TEM image of a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection modified with sodium alginate.
[0065] Figure 3Figure (a) shows the upconversion fluorescence spectra of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection with oleic acid ligand removed, the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection modified with sodium alginate, and the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection modified with sodium alginate after incubation with furazolidone; Figure (b) shows the upconversion fluorescence spectra of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection with oleic acid ligand removed, the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection modified with sodium alginate, and the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection modified with sodium alginate after incubation with malachite green.
[0066] Figure 4 Figure (a) shows the UV-Vis absorption spectra of furazolidone, malachite green, florfenicol, thiamphenicol, tobramycin, streptomycin, erythromycin, and amoxicillin. Figure (b) shows the ratiometric upconversion fluorescent nanoprobe modified with sodium alginate for veterinary drug detection provided in Example 1 of this application and its UV-Vis absorption spectra after incubation with furazolidone and malachite green.
[0067] Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 11 In the diagram, UCNPs-OA is an oleic acid-coated ratiometric upconversion fluorescent nanoprobe for veterinary drug detection; UCNPs is an oleic acid-ligand-removed ratiometric upconversion fluorescent nanoprobe for veterinary drug detection; ALG is sodium alginate; UCNPs-ALG is a sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection; FTD is furazolidone; MG is malachite green; FFC is florfenicol; THP is thiamphenicol; TOB is tobramycin; STR is streptomycin; ERY is erythromycin; and AMX is amoxicillin. UCNPs-ALG@FTD is a sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection incubated with furazolidone; and UCNPs-ALG@MG is a sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection incubated with malachite green. Detailed Implementation
[0068] This application provides a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection, its preparation method, and its application, in order to solve the technical problem of low reliability of fluorescence detection of veterinary drug residues in the prior art.
[0069] The technical solutions of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0070] Example 1
[0071] This embodiment provides a method for preparing a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection, including the preparation steps of the core rare earth source precursor solution, the preparation steps of the inner shell rare earth source precursor solution, the preparation steps of the middle shell rare earth source precursor solution, the preparation steps of the outer shell rare earth source precursor solution, the preparation steps of the core nanoparticles, the coating steps of the inner shell, the coating steps of the middle shell, the coating steps of the outer shell, the removal steps of the oleic acid ligand, and the modification steps of sodium alginate.
[0072] The preparation steps for the nuclear rare earth source precursor solution include:
[0073] 0.2 mmol of gadolinium acetate Gd(OAc)3, 0.196 mmol of ytterbium acetate Yb(OAc)3, 0.004 mmol of thulium acetate Tm(OAc)3, oleic acid (4 mL), and 1-octadecene (6 mL) were added to a two-necked flask; the mixture was heated to 160 °C in a heating mantle for 30 min to coordinate the reaction. The water in the reaction system was removed and the mixture was cooled to room temperature to obtain a nuclear rare earth source precursor solution.
[0074] The preparation steps for the inner shell rare earth source precursor solution include:
[0075] 0.4 mmol of ytterbium acetate (Yb(OAc)3), oleic acid (4 mL), and 1-octadecene (6 mL) were added to a two-necked flask; the mixture was heated to 160 °C in a heating mantle for 30 min to coordinate the reaction. The water in the reaction system was removed and the mixture was cooled to room temperature to obtain the inner shell rare earth source precursor solution.
[0076] The preparation steps for the intermediate shell rare earth source precursor solution include:
[0077] 0.2 mmol of gadolinium acetate Gd(OAc)3, 0.192 mmol of ytterbium acetate Yb(OAc)3, 0.008 mmol of erbium acetate Er(OAc)3, oleic acid (4 mL) and 1-octadecene (6 mL) were added to a two-necked flask; the mixture was heated to 160 °C in a heating mantle for 30 min to coordinate the reaction. The water in the reaction system was removed and the mixture was cooled to room temperature to obtain a middle-shell rare earth source precursor solution.
[0078] The preparation steps for the outer shell rare earth source precursor solution include:
[0079] 0.4 mmol of yttrium acetate Y(OAc)3, oleic acid (4 mL) and 1-octadecene (6 mL) were added to a two-necked flask; the mixture was heated to 160 °C in a heating mantle for 30 min, the water in the reaction system was removed, and the mixture was cooled to room temperature to obtain the outer shell rare earth source precursor solution.
[0080] The preparation steps of nuclear nanoparticles include:
[0081] 1.52 mmol of ammonium fluoride (NH4F) dissolved in 3.85 mL of methanol and 1 mmol of sodium hydroxide (NaOH) dissolved in 1 mL of methanol were added to the nuclear rare earth source precursor solution. The mixture was first heated to 50 °C and reacted for 30 min, then heated to 110 °C and reacted for 25 min. After removing the methanol, the mixture was evacuated for 10 min and then heated to 300 °C. The mixture was reacted for 1 h under an argon atmosphere to carry out a co-precipitation reaction. After the reaction was completed, the mixture was cooled to room temperature to obtain nuclear nanoparticles with the composition NaGdF4:Yb / Tm (Gd:Yb:Tm = 50 mol% : 49 mol% : 1 mol%), which were coated with oleic acid.
[0082] The oleic acid-coated core nanoparticles obtained from the coprecipitation reaction were stored in a cyclohexane dispersion for later use. The procedure was as follows: the oleic acid-coated core nanoparticles obtained from the coprecipitation reaction were transferred to a centrifuge tube along with the reaction solution. Anhydrous ethanol (4 mL, 99.5%) was added, the mixture was shaken and centrifuged at 7500 rpm for 6 min, and the supernatant was removed. Then, cyclohexane (4 mL, 99.5%) and anhydrous ethanol (8 mL, 99.5%) were added, the mixture was shaken and centrifuged at 7500 rpm for 6 min, and the supernatant was removed. Then, cyclohexane (4 mL, 99.5%), anhydrous ethanol (4 mL, 99.5%) and methanol (4 mL, 99.5%) were added, the mixture was shaken and centrifuged at 7500 rpm for 6 min, and the supernatant was removed. Finally, the obtained oleic acid-coated core nanoparticles were dispersed in a cyclohexane dispersion (4 mL, 99.5%), sealed, and stored at low temperature.
[0083] The inner shell encapsulation process includes:
[0084] Oleic acid-coated core nanoparticles (3.9 mL) in a cyclohexane dispersion, 1.52 mmol of ammonium fluoride (NH4F) dissolved in 3.85 mL of methanol, and 1 mmol of sodium hydroxide (NaOH) dissolved in 1 mL of methanol were added to the inner shell rare earth source precursor solution. The mixture was first heated to 50 °C and reacted for 30 min, then heated to 110 °C and reacted for 25 min. After removing the methanol, the mixture was evacuated for 10 min and then heated to 300 °C. The mixture was then reacted for 1 h under an argon atmosphere to carry out a co-precipitation reaction. After the reaction was completed, the mixture was cooled to room temperature to obtain core nanoparticles coated with an inner shell. The composition of the core nanoparticles was NaGdF4:Yb / Tm@NaYbF4, and the core nanoparticles were coated with oleic acid.
[0085] The oleic acid-coated NaGdF4:Yb / Tm@NaYbF4 nanoparticles obtained from the co-precipitation reaction were stored in a cyclohexane dispersion for later use. The procedure was as follows: the oleic acid-coated NaGdF4:Yb / Tm@NaYbF4 nanoparticles obtained from the co-precipitation reaction, along with the reaction solution, were transferred to a centrifuge tube. First, anhydrous ethanol (4 mL, 99.5%) was added, the mixture was shaken thoroughly, and centrifuged at 7500 rpm for 6 min. The supernatant was removed. Then, cyclohexane (4 mL, 99.5%) and anhydrous ethanol (8 mL, 99.5%) were added, the mixture was shaken thoroughly, and centrifuged at 7500 rpm for 6 min. The supernatant was removed. Finally, cyclohexane (4 mL, 99.5%), anhydrous ethanol (4 mL, 99.5%), and methanol (4 mL, 99.5%) were added, the mixture was shaken thoroughly, and centrifuged at 7500 rpm for 6 min. After removing the supernatant, the oleic acid-coated NaGdF4:Yb / Tm@NaYbF4 nanoparticles were dispersed in cyclohexane dispersion (3.9 mL, 99.5%) and stored in a sealed container at low temperature.
[0086] The encapsulation steps for the intermediate shell include:
[0087] NaGdF4:Yb / Tm@NaYbF4 nanoparticles (3.8 mL) coated with oleic acid in a cyclohexane dispersion, 1.52 mmol of ammonium fluoride (NH4F) dissolved in 3.85 mL of methanol, and 1 mmol of sodium hydroxide (NaOH) dissolved in 1 mL of methanol were added to the intermediate shell rare earth source precursor solution. The mixture was first heated to 50 °C and reacted for 30 min, then heated to 110 °C and reacted for 25 min. After removing the methanol, the mixture was evacuated for 10 min and then heated to 300 °C. The mixture was then reacted for 1 h under an argon atmosphere to carry out a co-precipitation reaction. After the reaction was completed, the mixture was cooled to room temperature to obtain core nanoparticles coated with an inner shell and an intermediate shell. The composition of these nanoparticles was NaGdF4:Yb / Tm@NaYbF4@NaGdF4:Yb / Er, and they were coated with oleic acid. The ratio of Gd:Yb:Er in NaGdF4:Yb / Er was 50 mol%:48 mol%:2 mol%).
[0088] Oleic acid-coated NaGdF4:Yb / Tm@NaYbF4@NaGdF4:Yb / Er nanoparticles were stored in a cyclohexane dispersion for later use. The process involved transferring the oleic acid-coated NaGdF4:Yb / Tm@NaYbF4@NaGdF4:Yb / Er nanoparticles obtained from the co-precipitation reaction, along with the reaction solution, to a centrifuge tube. First, anhydrous ethanol (4 mL, 99.5%) was added, the mixture was shaken thoroughly, and centrifuged at 7500 rpm for 6 min. The supernatant was removed. Then, cyclohexane (4 mL, 99.5%) and anhydrous ethanol (8 mL, 99.5%) were added, the mixture was shaken thoroughly, and centrifuged at 7500 rpm for 6 min. The supernatant was removed. Finally, cyclohexane (4 mL, 99.5%), anhydrous ethanol (4 mL, 99.5%), and methanol (4 mL, 99.5%) were added, the mixture was shaken thoroughly, and centrifuged at 7500 rpm for 6 min. After removing the supernatant, the oleic acid-coated NaGdF4:Yb / Tm@NaYbF4@NaGdF4:Yb / Er nanoparticles were dispersed in cyclohexane dispersion (3.8 mL, 99.5%), sealed, and stored at low temperature.
[0089] The outer shell coating process includes:
[0090] 3.7 mL of oleic acid-coated NaGdF4:Yb / Tm@NaYbF4@NaGdF4:Yb / Er core nanoparticles (from a cyclohexane dispersion), 1.52 mmol of ammonium fluoride (NH4F) dissolved in 3.85 mL of methanol, and 1 mmol of sodium hydroxide (NaOH) dissolved in 1 mL of methanol were added to the outer shell rare earth source precursor solution. The mixture was first heated to 50 °C and reacted for 30 min, then heated to 110 °C and reacted for 25 min. After removing the methanol, the mixture was evacuated for 10 min and then heated to 300 °C under an argon atmosphere for 1 minute. h was subjected to a co-precipitation reaction. After the reaction was completed and cooled to room temperature, core nanoparticles coated with an inner shell, a middle shell and an outer shell were obtained. The composition was NaGdF4:Yb / Tm@NaYbF4@NaGdF4:Yb / Er@NaYF4. They were coated with oleic acid, which is an oleic acid-coated ratiometric upconversion fluorescent nanoprobe for veterinary drug detection. The structure is a core-shell-shell-shell structure.
[0091] Oleic acid-coated ratiometric upconversion fluorescent nanoprobes for veterinary drug detection were stored in a cyclohexane dispersion for later use. The process involved transferring the oleic acid-coated ratiometric upconversion fluorescent nanoprobes for veterinary drug detection, obtained from the co-precipitation reaction, along with the reaction solution, to a centrifuge tube. First, anhydrous ethanol (4 mL, 99.5%) was added, the tube was shaken thoroughly, and centrifuged at 7500 rpm for 6 min. The supernatant was removed. Then, cyclohexane (4 mL, 99.5%) and anhydrous ethanol (8 mL, 99.5%) were added, the tube was shaken thoroughly, and centrifuged at 7500 rpm for 6 min. The supernatant was removed. Next, cyclohexane (4 mL, 99.5%), anhydrous ethanol (4 mL, 99.5%), and methanol (4 mL, 99.5%) were added, the tube was shaken thoroughly, and centrifuged at 7500 rpm for 6 min. The supernatant was removed. Finally, the oleic acid-coated ratiometric upconversion fluorescent nanoprobes for veterinary drug detection were dispersed in a cyclohexane dispersion (3.7 mL, 99.5%), sealed, and stored at low temperature.
[0092] The steps for removing oleic acid ligands include:
[0093] To avoid oleic acid affecting the modification of sodium alginate, the oleic acid ligand needs to be removed. The process involves dispersing 3.6 mL of oleic acid-coated ratiometric upconversion fluorescent nanoprobes for veterinary drug detection in a cyclohexane dispersion in 3.6 mL of a 0.1 M hydrochloric acid aqueous solution. After sonication at 30 °C for 1 h and centrifugation at 15,000 rpm for 30 min, the resulting particles are collected. These are the ratiometric upconversion fluorescent nanoprobes for veterinary drug detection with the oleic acid ligand removed. Subsequently, the ratiometric upconversion fluorescent nanoprobes for veterinary drug detection with the oleic acid ligand removed are washed 2-3 times with ultrapure water. Finally, the ratiometric upconversion fluorescent nanoprobes for veterinary drug detection with the oleic acid ligand removed are dispersed in 3.6 mL of ultrapure water and sealed for low-temperature storage.
[0094] The modification steps for sodium alginate include:
[0095] Dissolve 2.18 g of sodium acetate trihydrate powder in 100 mL of ultrapure water, then add 0.54 g of glacial acetic acid, mix well, and bring the volume to 250 mL. Mix well to prepare an acetate-sodium acetate buffer solution with pH=5.0. Mix 5 mg of sodium alginate with the prepared 10 mL acetate-sodium acetate buffer solution, shake and sonicate for 30 min to dissolve, and prepare a 0.5 mg / mL sodium alginate solution.
[0096] A ratiometric upconversion fluorescent nanoprobe for veterinary drug detection (0.1 mL) with oleic acid ligand removed was added to 0.04–0.0425 mL of sodium alginate solution and stirred at room temperature for 15 min. During stirring, the sodium alginate solution contained functional groups such as carboxyl and hydroxyl groups, which were electrostatically coated onto the outer shell surface of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection. A sodium alginate-modified ratiometric upconversion fluorescent nanoprobe solution with a concentration of approximately 2 mM was obtained. The sodium alginate modification improved the dispersibility of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection in aqueous medium.
[0097] Experimental Example 1
[0098] This experiment describes the structure and performance of the oleic acid-coated ratiometric upconversion fluorescent nanoprobes (UCNPs-OA) for veterinary drug detection, the oleic acid-ligand-removed ratiometric upconversion fluorescent nanoprobes (UCNPs) for veterinary drug detection, and the sodium alginate-modified ratiometric upconversion fluorescent nanoprobes (UCNPs-ALG) for veterinary drug detection provided in Example 1.
[0099] The instruments used for structural characterization and performance testing included: a Japanese HT7700 transmission electron microscope with an operating voltage of 100 kV; and a US Thermo-Filsher-Nicolet 6700 microscope with a scanning range of 4000–500 cm⁻¹. -1 The resolution is 1cm. -1 The number of scans was 32; the Ocean Optics USB2000+ fluorescence spectrometer used a 980 nm infrared semiconductor laser as the excitation source.
[0100] To investigate the success of oleic acid ligand removal and sodium alginate modification, this experiment characterized the oleic acid-coated ratiometric upconversion fluorescent nanoprobe for veterinary drug detection, the oleic acid-ligand-removed ratiometric upconversion fluorescent nanoprobe for veterinary drug detection, and the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection using infrared spectroscopy. Their infrared spectra are shown below. Figure 1 As shown; from Figure 1 It can be seen that oleic acid-coated ratiometric upconversion fluorescent nanoprobes for veterinary drug detection have… , Characteristic peaks exist at positions such as [positions not specified], while ratiometric upconversion fluorescent nanoprobes for veterinary drug detection, with oleic acid ligands removed, exhibit [specific characteristics]. , The absence of characteristic peaks at certain positions indicates that the oleic acid ligand was successfully removed; while sodium alginate (ALG) showed no characteristic peaks at other positions. , Characteristic peaks related to carboxyl and hydroxyl groups are present at positions such as [positions missing], while sodium alginate-modified ratiometric upconversion fluorescent nanoprobes for veterinary drug detection [missing information]. , The presence of characteristic peaks at certain positions indicates that the outer shell of the ratiometric upconversion fluorescent nanoprobe used for veterinary drug detection has been successfully coated and modified with sodium alginate, thereby improving the dispersion stability of the ratiometric upconversion fluorescent nanoprobe used for veterinary drug detection in aqueous media.
[0101] To investigate the microstructure of ratiometric upconversion fluorescent nanoprobes, this experiment performed electron microscopy analysis on the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1 (with oleic acid ligand removed) and the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection modified with sodium alginate. The electron micrographs are shown below. Figure 2 As shown in Figures (a) and (b); from Figure 2 As can be seen from Figures (a) and (b), the particle size of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection with oleic acid ligand removed and the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection modified with sodium alginate provided in Example 1 is between 40 and 50 nm.
[0102] To investigate the upconversion fluorescence performance of ratiometric upconversion fluorescent nanoprobes, this experiment tested the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1 (with oleic acid ligand removed) and the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection modified with sodium alginate. Their upconversion fluorescence spectra are shown below. Figure 3 As shown; from Figure 3 It can be seen that the upconversion fluorescence spectra of the ratiometric upconversion fluorescent nanoprobes for veterinary drug detection with the oleic acid ligand removed and those modified with sodium alginate for veterinary drug detection are basically the same, indicating that modifying sodium alginate does not affect its fluorescence properties. However, when furazolidone (FTD) or malachite green (MG) is added, the upconversion fluorescence spectrum changes, such as... Figure 3 As shown in Figure (a), after the addition of furazolidone, the upconversion emission intensity of the ratiometric upconversion fluorescent nanoprobe used for veterinary drug detection decreased significantly at 361 nm, exhibiting quenching, while the upconversion blue light emission intensity at 450 nm remained essentially unchanged, which can be used as an internal standard for upconversion fluorescence detection; Figure 3As shown in Figure (b), after the addition of malachite green, the upconversion emission intensity of the ratiometric upconversion fluorescent nanoprobe used for veterinary drug detection decreased significantly at 540 nm, resulting in quenching, while the upconversion NIR emission intensity at 800 nm remained essentially unchanged. This demonstrates that the ratiometric upconversion fluorescent nanoprobe provided in Example 1 of this application can quantitatively detect furazolidone or malachite green by measuring the ratio of fluorescence signal intensities at two wavelengths. Through a self-calibration mechanism, it effectively offsets the influence of non-specific factors such as environmental and instrument fluctuations on the fluorescence detection results, thereby improving the reliability of fluorescence detection of veterinary drug residues.
[0103] Experiment Example 2
[0104] This experimental example tests the performance of the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe (UCNPs-ALG) for veterinary drug detection provided in Example 1. The instruments used for performance testing include: a PerkinElmer Lambda 950 UV-Vis-NIR spectrophotometer with a scanning wavelength range of 200~800 nm; and a Marine Optics USB2000+ fluorescence spectrometer with a 980 nm infrared semiconductor laser as the excitation source.
[0105] To investigate the selectivity of ratiometric upconversion fluorescent nanoprobes, this experiment first tested the UV-Vis absorption spectra of aqueous solutions of furazolidone (FTD), malachite green (MG), florfenicol (FFC), thiamphenicol (THP), tobramycin (TOB), streptomycin (STR), erythromycin (ERY), and amoxicillin (AMX). Then, the UV-Vis absorption spectra of aqueous solutions of furazolidone (FTD) or malachite green (MG) at a concentration of 2.5 μM were compared with those of the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1. The results are as follows: Figure 4 As shown; from Figure 4 As shown in Figure (a), furazolidone (FTD) has broad absorption in the 200–450 nm range, while malachite green (MG) has broad absorption in the 200–700 nm range. Figure 4As shown in Figure (b), the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1 did not have an obvious absorption peak. However, after the addition of furazolidone or malachite green, the UV-Vis absorption spectrum showed the characteristic absorption of furazolidone or malachite green. This indicates that furazolidone has the ability to specifically absorb and upconvert fluorescence in the 200-450 nm range, while malachite green has the ability to specifically absorb and upconvert fluorescence in the 200-700 nm range. Therefore, the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1 has the potential to have good selectivity for furazolidone and malachite green.
[0106] After testing the UV-Vis absorption spectra, this experimental example incubated different veterinary drugs at equal concentrations (70 μM), including furazolidone (FTD), florfenicol (FFC), thiamphenicol (THP), tobramycin (TOB), streptomycin (STR), erythromycin (ERY), amoxicillin (AMX), and the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1, at room temperature for 5 min, and measured the upconversion fluorescence spectra. The ratiometric fluorescence results are as follows: Figure 5 As shown; and malachite green (MG), florfenicol (FFC), thiamphenicol (THP), tobramycin (TOB), streptomycin (STR), erythromycin (ERY), amoxicillin (AMX), and the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1 were incubated at room temperature for 5 min, and the upconversion fluorescence spectra were measured. The ratiometric fluorescence results are shown below. Figure 6 As shown; from Figure 5 and Figure 6 As can be seen, the ratiometric upconversion fluorescent nanoprobe modified with sodium alginate for veterinary drug detection provided in Example 1 remained essentially unchanged in intensity after incubation with florfenicol, thiamphenicol, tobramycin, streptomycin, erythromycin, amoxicillin, etc. However, the intensity of upconversion fluorescence was significantly quenched upon the addition of furazolidone or malachite green, indicating that the fluorescent probe has good selectivity and can be used for the detection of specific veterinary drugs furazolidone and malachite green.
[0107] Experimental Example 3
[0108] This experimental example tests the performance of the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe (UCNPs-ALG) for veterinary drug detection provided in Example 1. The instruments used for performance testing include: a scanning wavelength range of 200~800 nm; a Marine Optics USB2000+ fluorescence spectrometer with a 980 nm infrared semiconductor laser as the excitation source.
[0109] To investigate the sensitivity and quantitative detection capability of a ratiometric upconversion fluorescent nanoprobe for furazolidone (FTD), this experiment used the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1. Furazolidone at concentrations ranging from 0 to 100 µM was added, and the mixture was incubated at room temperature for 5 min. The concentration of furazolidone was detected by measuring the change in upconversion fluorescence intensity. The upconversion fluorescence spectra were obtained as follows: Figure 7 As shown; from Figure 7 As can be seen, with the gradual increase of furazolidone concentration, the fluorescence intensity of the sodium alginate-modified ratiomic upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1 gradually decreased at 361 nm, while the fluorescence intensity at 450 nm remained unchanged, which can be used as an internal standard. A scatter plot of the ratio of fluorescence intensity at 361 nm to that at 450 nm was also established, and the results are shown below. Figure 8 As shown, then... Figure 8 The results of performing local linear fitting on the 0–20 μM interval of the scatter plot of the data shown are as follows. Figure 9 As shown, for Figure 8 Local linear fitting was performed on the 30–80 μM interval of the scatter plot of the data shown, and the results are as follows. Figure 10 As shown; from Figure 9 and Figure 10 It can be seen that when the concentration of furazolidone is in the range of 0~20 μM or 30~80 μM, the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1 has a good linear response relationship with the furazolidone concentration, with linear equations of y=0.4777-0.0095x and R0, respectively. 2 =0.9972, y= 0.3198-0.0031 x, R 2 =0.9986; that is, after mixing and incubating furazolidone at a concentration of 0~80 μM with the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1, a linear relationship between the upconversion fluorescence intensity and the furazolidone concentration can be established. Then, after mixing and incubating the furazolidone to be detected with the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1, the concentration of the furazolidone to be detected can be quantitatively obtained by substituting the fluorescence intensity of the obtained ratiometric upconversion fluorescent nanoprobe into the linear relationship between the upconversion fluorescence intensity and the furazolidone concentration.
[0110] To further investigate the sensitivity and quantitative detection capability of ratiometric upconversion fluorescent nanoprobes for malachite green (MG), this experiment used the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1. Malachite green at concentrations ranging from 0 to 150 µM was added, and the mixture was incubated at room temperature for 5 min. The concentration of malachite green was detected by measuring changes in upconversion fluorescence intensity. The upconversion fluorescence spectra were obtained as follows: Figure 11 As shown; from Figure 11 It can be seen that as the concentration of malachite green gradually increases, the fluorescence intensity of the sodium alginate-modified ratiomic upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1 gradually decreases at 540 nm, while the fluorescence intensity at 800 nm remains unchanged, which can be used as an internal standard. A scatter plot of the ratio of fluorescence intensity at 540 nm to that at 800 nm was established, and the results are shown in Figure 12. Subsequently, [further details are needed]. Figure 12 The results of performing local linear fitting on the 0–40 μM interval of the scatter plot shown are as follows. Figure 13 As shown, for Figure 12 Local linear fitting was performed on the 40–90 μM interval of the scatter plot of the data shown, and the results are as follows. Figure 14 As shown; from Figure 13 and Figure 14 It can be seen that when the concentration of malachite green is in the range of 0~40 μM or 40~90 μM, the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1 has a good linear response relationship with the concentration of malachite green, and the linear equations are y = 1.8021 - 0.0231x, R0, and R0, respectively. 2 =0.9974, y= 1.0195-0.01033 x, R 2 =0.9990; that is, after mixing and incubating 0~90 μM concentration of malachite green and the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1, a linear relationship between the upconversion fluorescence intensity and the concentration of malachite green can be established. Then, after mixing and incubating the malachite green to be detected and the sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection provided in Example 1, the concentration of the malachite green to be detected can be quantitatively obtained by substituting the fluorescence intensity of the obtained ratiometric upconversion fluorescent nanoprobe into the linear relationship between the upconversion fluorescence intensity and the concentration of malachite green.
[0111] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.
Claims
1. A ratiometric upconversion fluorescent nanoprobe for veterinary drug detection, characterized in that, From the inside out, it includes: core nanoparticles, inner shell, middle shell, and outer shell; The inner shell encapsulates the core nanoparticles, the middle shell encapsulates the inner shell, and the outer shell encapsulates the middle shell. The chemical composition of the nuclear nanoparticles is: NaGdF4:Yb / Tm; The chemical composition of the inner shell is: NaYbF4; The chemical composition of the intermediate shell is: NaGdF4:Yb / Er; The chemical composition of the outer shell is NaYF4.
2. The ratiometric upconversion fluorescent nanoprobe for veterinary drug detection according to claim 1, characterized in that, In the nuclear nanoparticles, the molar ratio of Gd:Yb:Tm is 47~69.5:30~50:0.5~3; In the intermediate shell, the molar ratio of Gd:Yb:Er is 47~69.5:30~50:0.5~3.
3. The ratiometric upconversion fluorescent nanoprobe for veterinary drug detection according to claim 1, characterized in that, The particle size of the nuclear nanoparticles is 10~30 nm; The thickness of the inner shell is 3~10 nm; The thickness of the intermediate shell is 1~10 nm; The thickness of the outer shell is 3~10 nm.
4. The ratiometric upconversion fluorescent nanoprobe for veterinary drug detection according to claim 1, characterized in that, The outer shell of the ratiometric upconversion fluorescent nanoprobe used for veterinary drug detection is modified with sodium alginate.
5. A method for preparing a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection, characterized in that, The ratiometric upconversion fluorescent nanoprobe for veterinary drug detection as described in any one of claims 1-4 can be prepared by the following steps: Gadolinium salt, ytterbium salt, and thulium salt were dissolved in oleic acid and 1-octadecene to carry out a coordination reaction, thereby obtaining a nuclear rare earth source precursor solution. Ytterbium salt was dissolved in oleic acid and 1-octadecene for coordination reaction to obtain an inner shell rare earth source precursor solution; Gadolinium salt, ytterbium salt, and erbium salt were dissolved in oleic acid and 1-octadecene to carry out a coordination reaction, thereby obtaining a middle-shell rare earth source precursor solution. Yttrium salt was dissolved in oleic acid and 1-octadecene for a coordination reaction to obtain an outer shell rare earth source precursor solution; Alkaline fluorine source and alkaline sodium source were added to the nuclear rare earth source precursor solution for co-precipitation reaction to obtain nuclear nanoparticles; Alkaline fluorine source, alkaline sodium source and core nanoparticles are added to the inner shell rare earth source precursor solution for co-precipitation reaction to obtain core nanoparticles coated with inner shell. An alkaline fluorine source, an alkaline sodium source, and core nanoparticles coated with an inner shell were added to a rare earth source precursor solution with an intermediate shell for co-precipitation reaction to obtain core nanoparticles coated with an inner shell and an intermediate shell in sequence. An alkaline fluorine source, an alkaline sodium source, and core nanoparticles coating the inner and middle shells were added to the rare earth source precursor solution of the outer shell for co-precipitation reaction to obtain a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection.
6. The method for preparing a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection according to claim 5, characterized in that, It also includes the following steps: A ratiometric upconversion fluorescent nanoprobe for veterinary drug detection was added to a hydrochloric acid aqueous solution and subjected to sonication and centrifugation to obtain a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection with oleic acid ligand removed. A ratiometric upconversion fluorescent nanoprobe for veterinary drug detection with oleic acid ligands removed was added to a sodium alginate solution and stirred to obtain a sodium alginate-modified ratiometric upconversion fluorescent nanoprobe for veterinary drug detection.
7. The method for preparing a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection according to claim 5, characterized in that, The coordination reaction is carried out at a temperature of 150-180℃ for 20-40 minutes.
8. The method for preparing a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection according to claim 5, characterized in that, The coprecipitation reaction process includes: reacting sequentially at 40~60℃ for 20~40 min, at 100~120℃ for 15~40 min, and at 280~320℃ for 40~80 min under an argon atmosphere.
9. A method for preparing a ratiometric upconversion fluorescent nanoprobe for veterinary drug detection according to claim 5, characterized in that, The alkaline fluorine source is selected from ammonium fluoride, and the alkaline sodium source is selected from sodium hydroxide; The gadolinium salt is selected from at least one of acetate, nitrate, sulfate, and chloride; the ytterbium salt is selected from at least one of acetate, nitrate, sulfate, and chloride; the thulium salt is selected from at least one of acetate, nitrate, sulfate, and chloride; the erbium salt is selected from at least one of acetate, nitrate, sulfate, and chloride; and the yttrium salt is selected from at least one of acetate, nitrate, sulfate, and chloride.
10. The application of the ratiometric upconversion fluorescent nanoprobe for veterinary drug detection according to any one of claims 1-4 in the detection of furazolidone or malachite green.