A dual-mode fluorescent nanoprobe and its preparation method and application

Abstract

The present application belongs to the field of antibiotic detection, and in particular relates to a dual-mode fluorescent nanoprobe, a preparation method and an application thereof; the dual-mode fluorescent nanoprobe provided in the present application comprises, from the inside out, a core nanoparticle NaGdF4:Yb / Tm, a NaYF4:Tb inner shell, a NaGdF4:Ce middle shell and a NaYF4 outer shell; the dual-mode fluorescent nanoprobe can be excited by a 980 nm laser or a 250 nm light source to produce a fluorescence emission spectrum, and combines up-conversion nanomaterials and down-conversion nanomaterials to detect antibiotics; and the detection results have good sensitivity, accuracy, selectivity and anti-interference ability, thereby solving the technical problem of limitations of single-mode fluorescent probes in the prior art.

Description

Technical Field

[0001] The present application belongs to the field of antibiotic detection, and in particular relates to a dual-mode fluorescent nanoprobe and its preparation method and application. Background Art

[0002] Antibiotics are used in the treatment and prevention of diseases in areas such as animal husbandry, agriculture, and human health. However, most antibiotics are difficult to fully utilize and may remain in animals and the environment, entering the human body through the food chain, posing a potential hazard to human health. Therefore, it is necessary to test antibiotic residues in food and the environment to ensure that they are within a safe range and do not pose a potential hazard to human health.

[0003] Currently, antibiotics can be detected using methods such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography tandem mass spectrometry (LC-MS / MS), spectrophotometry, and enzyme-linked immunosorbent assay (ELISA). Current antibiotic detection methods, such as HPLC, LC-MS, and LC-MS / MS, require relatively expensive instruments and training for experimental operators, and the experimental detection is time-consuming. Compared with these traditional antibiotic detection methods, the fluorescence spectrometer used in fluorescence detection is relatively inexpensive, sample pretreatment and detection operations are relatively simple, and it can also achieve qualitative and quantitative detection of samples, making it a detection method more suitable for practical applications.

[0004] In addition to commonly used fluorescent probes constructed from organic dyes such as cyanine dyes and quantum dots such as carbon quantum dots, upconversion nanomaterials can also be used as fluorescent probes using anti-Stokes luminescence. Upconversion nanomaterials can convert near-infrared excitation light into high-energy fluorescence in the ultraviolet or visible range. Upon encountering antibiotics, if the antibiotic's absorption spectrum overlaps with the fluorescence emission spectrum of the upconversion nanomaterial, the fluorescence intensity is significantly quenched. Furthermore, as the concentration of the added antibiotic increases, the fluorescence intensity of the upconversion fluorescent nanoprobe gradually decreases. This allows for qualitative and quantitative detection of antibiotics based on the correlation between antibiotic concentration and the change in upconversion luminescence intensity. Lanthanide ion-doped nanoparticles (RENPs) possess unique optical properties. Irradiation with a near-infrared 980 nm laser effectively eliminates background fluorescence interference while emitting light at a lower wavelength. Compared to traditional fluorescent reagents, RENPs offer advantages such as high chemical stability, low toxicity, high quantum yield, narrow emission peak, and long fluorescence lifetime. Therefore, dual-mode fluorescent nanoprobes based on RENPs, combining the advantages of both upconversion and downconversion fluorescence sensing, offer broad application prospects. Summary of the Invention

[0005] In view of this, the present application provides a dual-mode fluorescent nanoprobe and a preparation method and application thereof, which are used to solve the technical problem of limitations of single-mode fluorescent probes in the prior art.

[0006] In a first aspect, the present application provides a dual-mode fluorescent nanoprobe comprising a core nanoparticle NaGdF4:Yb / Tm, a NaYF4:Tb inner shell, a NaGdF4:Ce middle shell, and a NaYF4 outer shell;

[0007] The NaYF4:Tb inner shell layer covers the core nanoparticle NaGdF4:Yb / Tm;

[0008] The NaGdF4:Ce intermediate shell layer covers the NaYF4:Tb inner shell layer;

[0009] The NaYF4 outer shell layer covers the NaGdF4:Ce middle shell layer.

[0010] Preferably, the particle size of the dual-mode fluorescent nanoprobe is 40-80 nm.

[0011] Preferably, the particle size of the core nanoparticles NaYbF4:Tm / Gd is 20-25 nm;

[0012] The thickness of the NaYF4:Tb inner shell is 7-20 nm;

[0013] The thickness of the NaGdF4:Ce intermediate shell is 7-20 nm;

[0014] The thickness of the NaYF4 outer layer is 7-20 nm.

[0015] Preferably, in the core nanoparticles NaGdF4:Yb / Tm, the molar ratio of Yb: Tm: Gd is 40-80:0.5-2:20-60;

[0016] In the NaYF4:Tb inner shell, the molar ratio of Y:Tb is 70-95:5-30;

[0017] In the NaGdF4:Ce intermediate shell layer, the molar ratio of Gd: Ce is 70-90:10-20.

[0018] Preferably, in the core nanoparticles NaGdF4:Yb / Tm, the molar ratio of Yb: Tm: Gd is 49:1:50;

[0019] In the NaYF4:Tb inner shell, the molar ratio of Y:Tb is 85:15;

[0020] In the NaGdF4:Ce intermediate shell layer, the molar ratio of Gd:Ce is 85:15.

[0021] Preferably, in the dual-mode fluorescent nanoprobe, the NaYF4 outer shell is modified with amino-silica.

[0022] The second aspect of the present application provides a method for preparing a dual-mode fluorescent nanoprobe, which can be used to prepare the dual-mode fluorescent nanoprobe described in the first aspect. The preparation method comprises the following steps:

[0023] Step S1, adding ammonium fluoride and sodium hydroxide to a precursor reaction solution containing ytterbium / thulium / gadolinium-oleic acid complex to perform a core co-precipitation reaction to obtain core nanoparticles NaGdF4:Yb / Tm;

[0024] Step S2, adding core nanoparticles NaGdF4:Yb / Tm, ammonium fluoride, and sodium hydroxide to a precursor reaction solution containing a yttrium / terbium-oleic acid complex to carry out an inner shell co-precipitation reaction to prepare a core-shell structured NaGdF4:Yb / Tm@NaYF4:Tb;

[0025] Step S3, adding NaGdF4:Yb / Tm@NaYF4:Tb, ammonium fluoride, and sodium hydroxide to a precursor reaction solution containing a gadolinium / cerium-oleic acid complex to perform an intermediate shell co-precipitation reaction to prepare a core-shell-shell structured NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce;

[0026] Step S4, adding NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce, ammonium fluoride, and sodium hydroxide to the precursor reaction solution containing the yttrium-oleic acid complex to carry out an outer shell co-precipitation reaction to prepare a core-shell-shell-shell-shell dual-mode fluorescent nanoprobe.

[0027] Preferably, after step S4, the method further comprises the following steps:

[0028] Step S5, dispersing the dual-mode fluorescent nanoprobe in an ethanol solvent, adding ethyl orthosilicate in an alkaline environment and hydrolyzing it to obtain a silica-modified dual-mode fluorescent nanoprobe;

[0029] Step S6: adding 3-aminopropyltriethoxysilane to the silica-modified dual-mode fluorescent nanoprobe to perform an amination reaction to obtain the amination-silica-modified dual-mode fluorescent nanoprobe.

[0030] Preferably, in step S1, the method for preparing the precursor reaction solution of the ytterbium / thulium / gadolinium-oleic acid complex comprises: conducting a coordination reaction between ytterbium salt, thulium salt, gadolinium salt, oleic acid and 1-octadecene to prepare the precursor reaction solution of the ytterbium / thulium / gadolinium-oleic acid complex;

[0031] In step S2, the method for preparing the precursor reaction solution of the yttrium / terbium-oleic acid complex comprises: conducting a coordination reaction between yttrium salt, terbium salt, oleic acid and 1-octadecene to prepare the precursor reaction solution of the yttrium / europium-oleic acid complex;

[0032] In step S3, the method for preparing the precursor reaction solution of the gadolinium / cerium-oleic acid complex comprises: conducting a coordination reaction between gadolinium salt, cerium salt, oleic acid, and 1-octadecene to prepare the precursor reaction solution of the gadolinium / cerium-oleic acid complex;

[0033] In step S4, the method for preparing the precursor reaction solution of the yttrium-oleic acid complex comprises: conducting a coordination reaction between yttrium salt, oleic acid and 1-octadecene to prepare the precursor reaction solution of the yttrium-oleic acid complex.

[0034] Preferably, in steps S1 to S4, the coordination reaction temperature is 150-180° C., and the time is 20-40 min.

[0035] Preferably, in step S1, the process of the nuclear coprecipitation reaction is: heating to 40-60°C for reaction for 20-40 min, heating to 100-120°C for reaction for 20-30 min, removing methanol, vacuuming for 5-15 min, then heating to 250-300°C, and reacting under argon atmosphere for 0.5-1.5 h;

[0036] In step S2, the inner shell co-precipitation reaction process is as follows: heating to 40-60 ° C for reaction for 20-40 min, heating to 100-120 ° C for reaction for 20-30 min, removing methanol, vacuuming for 5-15 min, then heating to 250-300 ° C, and reacting under argon atmosphere for 0.5-1.5 h;

[0037] In step S3, the process of the intermediate shell co-precipitation reaction is: heating to 40-60°C for reaction for 20-40 min, heating to 100-120°C for reaction for 20-30 min, removing methanol, vacuuming for 5-15 min, then heating to 250-300°C, and reacting under argon atmosphere for 0.5-1.5 h;

[0038] In step S4, the outer shell co-precipitation reaction process is: heating to 40-60 ° C for reaction for 20-40 min, heating to 100-120 ° C for reaction for 20-30 min, removing methanol, vacuuming for 5-15 min, then heating to 250-300 ° C, and reacting under argon atmosphere for 0.5-1.5 h.

[0039] Preferably, in step S5, the temperature for hydrolysis of ethyl orthosilicate is 35-45° C. and the time is 4-8 h;

[0040] In step S6, the amination reaction temperature is 35-45°C and the time is 4-8 hours;

[0041] The third aspect of the present application provides the use of a dual-mode fluorescent nanoprobe in detecting antibiotics.

[0042] The application is specifically: application in the detection of antibiotic pollution in water bodies.

[0043] Preferably, the antibiotic is selected from furazolidone.

[0044] Preferably, the application process comprises the steps of:

[0045] Step 1: mixing the dual-mode fluorescent nanoprobe with a concentration gradient antibiotic standard solution and performing fluorescence detection to establish a linear relationship between the concentration gradient antibiotic standard solution and the emission intensity of the dual-mode fluorescent nanoprobe;

[0046] Step 2: mixing the dual-mode fluorescent nanoprobe and the antibiotic sample to be detected, and then performing fluorescence detection to obtain the emission intensity of the dual-mode fluorescent nanoprobe to be detected;

[0047] Step 3: Substitute the emission intensity of the dual-mode fluorescent nanoprobe into the linear relationship between the concentration gradient of the antibiotic standard solution and the emission intensity of the dual-mode fluorescent nanoprobe to calculate the concentration of the antibiotic in the antibiotic sample to be detected.

[0048] In summary, the present application provides a dual-mode fluorescent nanoprobe, a preparation method and an application. The dual-mode fluorescent nanoprobe of the present application includes a core nanoparticle NaGdF4:Yb / Tm, a NaYF4:Tb inner shell, a NaGdF4:Ce middle shell and a NaYF4 outer shell from the inside to the outside; wherein, the dual-mode fluorescent nanoprobe can not only be used as an up-conversion nanomaterial to be excited by a 980 nm laser to produce a fluorescence emission spectrum, but can also be used as a down-conversion nanomaterial to be excited by a 250 nm light source to produce a fluorescence emission spectrum. According to actual conditions, a near-infrared semiconductor laser can be selected as the excitation light source for up-conversion fluorescence detection, or a xenon lamp can be used as the excitation light source to produce a fluorescence emission spectrum for qualitative and quantitative detection of antibiotics; at the same time, since the dual-mode fluorescent nanoprobe combines the advantages of up-conversion nanomaterials and down-conversion nanomaterials, it is beneficial to expand the detection concentration range of the fluorescent nanoprobe and improve the detection sensitivity; it can also select an emission peak with a better linear relationship or more sensitivity to the antibiotic concentration according to the fluorescence emission spectrum, thereby improving the accuracy and sensitivity of the fluorescent nanoprobe, thereby solving the technical problem of limitations of single-mode fluorescent probes in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0050] Figure 1 Figures showing the structure and performance test results of the core nanoparticles NaGdF4:Yb / Tm, core-shell nanoparticles NaGdF4:Yb / Tm @NaYF4:Tb, core-shell-shell nanoparticles NaYbF4:Tm / Gd@NaYF4:Tb@NaGdF4:Ce, and core-shell-shell-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 provided in Example 1 of the present application;

[0051] Figure 2 The UV-visible absorption spectra of NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 modified with amino-silica provided in Example 1 of the present application are added to various antibiotics and the antibiotic furazolidone;

[0052] Figure 3 This is a diagram showing the structure and performance test results of the amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 provided in Example 1;

[0053] Figure 4 The upconversion fluorescence spectra of NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 modified with amino-silica after adding different concentrations of the antibiotic furazolidone and the linear relationship between the concentration of the antibiotic furazolidone and the upconversion emission intensity are shown;

[0054] Figure 5 The down-conversion fluorescence spectra of NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 modified with amino-silica after adding different concentrations of the antibiotic furazolidone and the linear relationship between the concentration of the antibiotic furazolidone and the down-conversion emission intensity are shown;

[0055] Figure 6 The relative intensity graphs of up-conversion emission and down-conversion emission of amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 after adding an equal concentration of different antibiotics respectively;

[0056] Figure 7 Relative intensity of up-conversion emission and down-conversion emission of amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 after adding the antibiotic furazolidone and another antibiotic simultaneously.

[0057] Figure 1-7 In the figure, Core, Core-shell, Core-shell-shell, and Core-shell-shell-shell are respectively: core nanoparticles NaGdF4:Yb / Tm, core-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb, core-shell-shell nanoparticles NaYbF4:Tm / Gd@NaYF4:TbN@aGdF4:Ce, and core-shell-shell-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4;

[0058] ERY, STM, TOB, FFC, THP, AMX, and FZD correspond to erythromycin, streptomycin, tobramycin, florfenicol, thiamphenicol, amoxicillin, and furazolidone, respectively;

[0059] The composition of RENPs corresponds to: NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4,

[0060] The composition of RENPs@SiO2 corresponds to: amino-silica-modified NaGdF4:Yb / Tm / Gd@NaYF4:Tb@NaGdF4:Ce@NaYF4. DETAILED DESCRIPTION

[0061] The present application provides a dual-mode fluorescent nanoprobe and a preparation method and application thereof, which are used to solve the technical problem of limitations of single-mode fluorescent probes in the prior art.

[0062] The following will clearly and completely describe the technical solution of this application in conjunction with the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of this application.

[0063] In view of the defects of the current use of single-mode fluorescent probes such as up-conversion nanomaterials, the present application provides a dual-mode fluorescent nanoprobe, which is composed of core nanoparticles NaGdF4:Yb / Tm, NaYF4:Tb inner shell, NaGdF4:Ce middle shell and NaYF4 outer shell from the inside out; the dual-mode fluorescent nanoprobe can use a 980 nm near-infrared semiconductor laser as an excitation light source to produce an up-conversion fluorescence emission spectrum, and the emission intensity appears at positions such as 360 nm in the spectrum, or use a 250 nm xenon lamp as an excitation light source to produce a down-conversion fluorescence emission spectrum, and the emission intensity appears at positions such as 544 nm in the spectrum, and as the concentration of the added antibiotics such as furazolidone gradually increases, the up-conversion emission intensity at positions such as 360 nm will gradually decrease or increase. The down-conversion emission intensity at positions such as nm will gradually decrease and show a linear relationship with the concentration of antibiotics such as furazolidone. Therefore, the advantages of up-conversion nanomaterials and down-conversion nanomaterials can be combined to detect antibiotics. Compared with traditional single-mode fluorescent probes such as down-conversion nanomaterials, the detection concentration range of fluorescent nanoprobes for antibiotics can be expanded; the sensitivity and accuracy of fluorescent nanoprobes can be improved; and the defects of single-mode fluorescent probes in the existing technology can be solved.

[0064] Preferably, the dual-mode fluorescent nanoprobe provided in the present application is also modified with amino-silica on its surface, which is beneficial to improve its hydrophilicity and is suitable for the detection of antibiotics in water bodies.

[0065] Preferably, the size of the dual-mode fluorescent nanoprobe provided in the present application is 50-60 nm, the particle size of the core nanoparticle NaGdF4:Yb / Tm is 20-25 nm, the thickness of the NaYF4:Tb inner shell is 8-12 nm, the thickness of the NaGdF4:Ce middle shell is 8-12 nm, and the thickness of the NaYF4 outer shell is 10-15 nm;

[0066] At the same time, for the ratio of each element in the dual-mode fluorescent nanoprobe provided in this application, the molar ratio of Yb: Tm: Gd in the core nanoparticles NaYbF4:Tm / Gd is 40~80:0.5~2:20~60; in the NaYF4:Tb inner shell, the molar ratio of Y: Tb is 70~95:5~30, and in the NaGdF4:Ce intermediate shell, the molar ratio of Gd: Ce is 70~90:10~20.

[0067] The dual-mode fluorescent nanoprobe provided in the present application can obtain a fluorescence emission spectrum with better anti-Stokes luminescence / Stokes luminescence effects by adjusting its particle size and composition.

[0068] For the preparation method of the dual-mode fluorescent nanoprobe provided in this application, a rare earth-oleic acid complex solution can be prepared first, and then ammonium fluoride, sodium hydroxide and the rare earth-oleic acid complex can be subjected to a co-precipitation reaction to obtain the dual-mode fluorescent nanoprobe; during the preparation process, the core nanoparticles NaGdF4:Yb / Tm of the dual-mode fluorescent nanoprobe and other intermediate products can be stored in a cyclohexane solution.

[0069] Preferably, for the dual-mode fluorescent nanoprobe provided in the present application for detecting antibiotics, since furazolidone has a wide absorption in the range of 200-450 nm, the emission spectrum overlaps with a high region of the up-conversion nanomaterial, and at the same time overlaps with the excitation spectrum of the down-conversion nanomaterial.

[0070] Example 1

[0071] Example 1 of the present application provides a method for preparing a dual-mode fluorescent nanoprobe, which includes the steps of preparing a precursor reaction solution of a rare earth-oleic acid complex, a coprecipitation reaction, and an amino-silica modification step.

[0072] The steps of preparing the precursor reaction solution of the rare earth-oleic acid complex include:

[0073] Preparation of ytterbium / thulium / gadolinium-oleic acid complex precursor reaction solution: Ytterbium acetate (0.98 mL, 0.2 mol / L), thulium acetate (0.08 mL, 0.05 mol / L), gadolinium acetate (1 mL, 0.2 mol / L), 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 and reacted for 30 min. The water in the reaction system was removed and the reaction system was cooled to room temperature to obtain the ytterbium / thulium / gadolinium-oleic acid complex precursor reaction solution.

[0074] Preparation of yttrium / terbium-oleic acid complex precursor reaction solution: Yttrium acetate (1.7 mL, 0.2 mol / L), terbium acetate (1.2 mL, 0.05 mol / L), 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 and reacted for 30 min. The water in the reaction system was removed and the reaction mixture was cooled to room temperature to obtain a yttrium / terbium-oleic acid complex precursor reaction solution.

[0075] Preparation of the gadolinium / cerium-oleic acid complex precursor reaction solution: Gadolinium acetate (1.7 mL, 0.2 mol / L), cerium acetate (0.3 mL, 0.2 mol / L), 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 and reacted for 30 min. The water in the reaction system was removed and the mixture was cooled to room temperature to obtain the gadolinium / cerium-oleic acid complex precursor reaction solution.

[0076] Preparation of yttrium-oleic acid complex precursor reaction solution: Yttrium acetate (2 mL, 0.2 mol / L), oleic acid (4 mL) and 1-octadecene (6 mL) were added to a two-necked flask, heated to 160 °C in a heating mantle and reacted for 30 min. Water in the reaction system was removed and the reaction system was cooled to room temperature to obtain a yttrium-oleic acid complex precursor reaction solution.

[0077] The steps of the coprecipitation reaction include:

[0078] To the prepared ytterbium / thulium / gadolinium-oleic acid complex precursor reaction solution, NH4F4 (3.85 mL, 0.4 mol / L) and NaOH (1 mL, 1 mol / L) were added. The mixture was heated to 50°C for 30 min and then heated to 110°C for 25 min to remove methanol. After evacuation for 10 min, the temperature was raised to 300°C and the reaction was continued under argon atmosphere for 1 h to obtain an oleic acid-coated core nanoparticle reaction solution with a composition of NaGdF4:Yb / Tm (49 mol%:1 mol%). The core nanoparticles were washed and dispersed in 4 mL of cyclohexane.

[0079] To the prepared yttrium / terbium-oleic acid complex precursor reaction solution, 3.9 mL of oleic acid-coated core nanoparticle reaction solution, NH4F (3.85 mL, 0.4 mol / L), and NaOH (1 mL, 1 mol / L) were added. The mixture was heated to 50°C for 30 min and then heated to 110°C for 25 min to remove methanol. After evacuation for 10 min, the temperature was raised to 300°C and the reaction was continued under argon atmosphere for 1 h to obtain oleic acid-coated core-shell nanoparticle reaction solution. The core-shell nanoparticles had a composition of NaGdF4:Yb / Tm (49 mol%:1 mol%)@NaYF4:Tb (15 mol%). After washing, the mixture was dispersed in 6 mL of cyclohexane.

[0080] To the prepared gadolinium / cerium oleate complex precursor reaction solution, 3 mL of core-shell nanoparticle reaction solution, NH4F (3.85 mL, 0.4 mol / L), and NaOH (1 mL, 1 mol / L) were added. The mixture was heated to 50°C for 30 min and then heated to 110°C for 25 min to remove methanol. After evacuation for 10 min, the temperature was raised to 300°C and reacted under argon atmosphere for 1 h to obtain oleic acid-coated core-shell-shell nanoparticle reaction solution. The core-shell-shell nanoparticles had a composition of NaGdF4:Yb / Tm (49 mol%:1 mol%)@NaYF4:Tb(15 mol%)@NaGdF4:Ce(15 mol%). After washing, the mixture was dispersed in 3.8 mL of cyclohexane.

[0081] To the prepared yttrium-oleic acid complex precursor reaction solution, 3.6 mL of core-shell-shell nanoparticle reaction solution, NH4F (3.85 mL, 0.4 mol / L) and NaOH (1 mL, 1 mol / L) were added. The mixture was heated to 50 ℃ for 30 min and then heated to 110 ℃ for 25 min to remove methanol. After vacuuming for 10 min, the temperature was raised to 300 ℃ and reacted under argon atmosphere for 1 h to obtain oleic acid-coated core-shell-shell-shell nanoparticle reaction solution. The core-shell-shell-shell nanoparticles had a composition of NaGdF4:Yb / Tm (49 mol%:1 mol%)@NaYF4:Tb (15 mol%)@NaGdF4:Ce (15 mol%)@NaYF4. After washing, they were dispersed in 3.6 mL of cyclohexane.

[0082] The washing and storage process of the prepared core nanoparticles NaGdF4:Yb / Tm, core-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb, core-shell-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce, and core-shell-shell-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 includes:

[0083] The reaction products of the coprecipitation reaction, core nanoparticles NaGdF4:Yb / Tm, core-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb, core-shell-shell nanoparticles NaYbF4:Tm / Gd@NaYF4:Tb@NaGdF4:Ce and core-shell-shell-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4, were transferred to a centrifuge tube, and anhydrous ethanol (4 mL, 99.55%) was added, the suspension was evenly shaken, and the mixture was centrifuged at 7500 rpm for 6 min, and the supernatant liquid was removed; cyclohexane (4 mL, 99.5%) was added, anhydrous ethanol (8 mL, 99.5%) was added, the suspension was evenly shaken, and the mixture was centrifuged at 7500 rpm for 6 min, and the supernatant liquid was removed; cyclohexane (4 mL, 99.5%), anhydrous ethanol (4 mL, 99.5%), methanol (4 mL, 99.5%) were added, the suspension was evenly shaken, and the mixture was centrifuged at 7500 rpm for 6 min, and the supernatant liquid was removed; mL, 99.5%), suspended and shaken evenly, centrifuged at 7500 rpm for 6 min, removed the upper liquid, dispersed it in cyclohexane (99.5%), sealed and stored at low temperature to obtain a sample.

[0084] The steps of aminated silica modification include:

[0085] 4 mL of anhydrous ethanol was added to the core-shell-shell-shell nanoparticle reaction solution (2 mL of cyclohexane), and the mixture was centrifuged at 7500 rpm for 6 min to separate the core-shell-shell-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 from the cyclohexane solution. 40 mL of anhydrous ethanol was added, and the mixture was ultrasonically dispersed. The mixture was transferred to a two-necked flask and ultrasonicated for 45 min to disperse the mixture. 14 mL of water and 1.7 mL of ammonia water were added, and after stabilization in an oil bath at 40 °C, 40 μL of tetraethyl orthosilicate was slowly added, and the mixture was stirred at constant temperature for 6 h to obtain silica-modified core-shell-shell-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4.

[0086] Slowly add 70 μL of 3-aminopropyltriethoxysilane and stir at 40 °C for 6 h to obtain amino-silica-modified core-shell-shell-shell nanoparticles NaYbF4:Tm / Gd@NaYF4:Tb@NaGdF4:Ce@NaYF4; wash twice with anhydrous ethanol and store in 20 mL of anhydrous ethanol.

[0087] Experimental Example 1

[0088] This application conducts structural and performance tests on the core nanoparticles NaGdF4:Yb / Tm, core-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb, NaYbF4:Tm / Gd@NaYF4:Tb@NaGdF4:Ce, core-shell-shell-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4, and amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 provided in Example 1.

[0089] The instruments used for structural testing include a Japanese HT7700 transmission electron microscope with an operating voltage of 100 kV and an American Thermo-Filsher-Nicolet 6700 Fourier transform infrared spectrometer with a scanning range of 4000-500 cm -1 , with a resolution of 2 cm -1 , the number of scans was 32; the American PerkinElmer Lambda 950 UV-visible-near-infrared spectrophotometer, the scanning wavelength range was 200~800 nm;

[0090] The instruments used for performance testing include an Ocean Optics USB-2000+ fluorescence spectrometer, which uses a 980 nm near-infrared semiconductor laser as the excitation light source; and a US HORIBA Jobin Yvon fluorescence spectrometer, which uses a 250 nm xenon lamp as the excitation light source.

[0091] Among them, the transmission electron microscopy images of core nanoparticles NaGdF4:Yb / Tm, core-shell nanoparticles NaGdF4:Yb / Tm @NaYF4:Tb, core-shell-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce and core-shell-shell-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 are shown in Figure 2. Figure 1 As shown in Figures a~d in the figure, it can be seen from Figures a~d that the particle sizes are 22.3 nm, 32.4 nm, 41.7 and 55 nm respectively; and the up-conversion fluorescence spectrum obtained by using a 980 nm near-infrared semiconductor laser as the excitation light source and the down-conversion fluorescence spectrum obtained by using a 250 nm xenon lamp as the excitation light source are shown in Figures Figure 1 As shown in the e~f figures in the figure, it can be seen from the e~f figures that the core-shell-shell-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 can produce anti-Stokes luminescence / Stokes luminescence, which can combine the advantages of upconversion nanomaterials and downconversion nanomaterials to detect antibiotics.

[0092] The UV-visible absorption spectra of various antibiotics and furazolidone and the UV-visible absorption spectra of the aminated silica modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 provided in Example 1 of the present application are shown in FIG. Figure 2 As shown in Figures a~b in the figure, it can be seen from Figures a~b that the antibiotic furazolidone has a wide absorption spectrum; the amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 does not show a characteristic absorption peak, but when the furazolidone antibiotic is added, the characteristic absorption peak of furazolidone appears.

[0093] Transmission electron microscopy and infrared spectrum of amination-silica modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 Figure 3 As shown in Figures a~b, it can be seen from Figures a~b that the particle size is slightly larger, 57 nm; compared with the FT-IR spectrum of RENPs coated with oleic acid ligands (RENPs-OA), RENPs@SiO2 has a peak at 1068 cm -1 and 792 cm -1The characteristic peak of Si-O-Si appears at 457 cm -1 The characteristic peak at 1631 cm −1 The characteristic peaks appearing at come from the NH groups, indicating that the NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 surface is successfully modified by aminosilica.

[0094] The up-conversion fluorescence spectrum and furazolidone absorption spectrum overlay of amino-silica modified NaYbF4:Tm / Gd@NaYF4:Tb@NaGdF4:Ce@NaYF4, the down-conversion fluorescence excitation spectrum and furazolidone absorption spectrum overlay are shown in Figure 2. Figure 3 As shown in Figures c~d in the figure, it can be seen from Figures c~d that there is an overlapping area between the up-conversion fluorescence emission spectrum / down-conversion fluorescence excitation spectrum of amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 and the absorption spectrum of furazolidone, which has the possibility of fluorescence detection.

[0095] The up-conversion fluorescence spectra of NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4, amination-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 and after adding furazolidone, the down-conversion fluorescence spectra are as follows: Figure 3 As shown in the e~f figures in the figure, it can be seen from the e~f figures that after the addition of furazolidone, the emission intensity at positions such as 344 nm and 360 nm in the up-conversion fluorescence spectrum decreases, and the emission intensity at positions such as 544 nm in the down-conversion fluorescence spectrum decreases. This shows that those skilled in the art can use amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 to detect antibiotics such as furazolidone.

[0096] Experimental Example 2

[0097] This application conducts performance tests on the amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 provided in Example 1.

[0098] Among them, the instruments used for performance testing include the Ocean Optics USB-2000+ fluorescence spectrometer, which uses a 980 nm near-infrared semiconductor laser as the excitation light source; and the American HORIBA Jobin Yvon fluorescence spectrometer, which uses a xenon lamp as the excitation light source.

[0099] The results of the performance test are as follows Figure 4-5As shown in the figure, the test results obtained using a 980 nm near-infrared semiconductor laser as the excitation light source are as follows: Figure 4 As shown in the figure, with the gradual increase of furazolidone concentration (0~300 uM), the up-conversion emission intensity of amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 at 344 nm and 360 nm gradually decreased, and when the furazolidone concentration was in the range of 0.05~30 μM, the up-conversion emission relative intensity had a good linear relationship with the furazolidone concentration, and the linear equation was y = 0.441 - 0.0051 x; when the furazolidone concentration was in the range of 30~300 μM, the up-conversion emission relative intensity had a good linear relationship with the furazolidone concentration, and the linear equation was y = 0.3056 -0.0008 x, which indicated that when the furazolidone concentration was 0.05~300 When the concentration of furazolidone is 0.05-300 μM, the upconversion fluorescence intensity of the amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 is linearly related to the concentration of furazolidone, that is, after the present invention mixes and incubates furazolidone with a concentration gradient of 0.05-300 μM and the amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4, a linear relationship between the upconversion fluorescence intensity and the concentration of furazolidone can be established; then, after the furazolidone to be detected is mixed and incubated with the amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4, the upconversion emission intensity of the obtained amino-silica-modified NaYbF4:Tm / Gd@NaYF4:Tb@NaGdF4:Ce@NaYF4 is substituted into the linear relationship between the upconversion fluorescence intensity and the concentration of furazolidone to obtain the concentration of the furazolidone to be detected;

[0100] The test results are as follows: Figure 5 As shown, from Figure 5It can be seen that with the gradual increase of furazolidone concentration (0~300 uM), the down-conversion emission intensities of amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 at 488 nm, 544 nm, 583 nm and 621 nm gradually decrease, and when the furazolidone concentration is in the range of 1~50 μM, the relative intensity of the down-conversion emission has a good linear relationship with the furazolidone concentration, and the linear equation is y = 0.9762 - 0.0083 x; while when the furazolidone concentration is in the range of 60~300 μM, the relative intensity of the down-conversion emission has a good linear relationship with the furazolidone concentration, and the linear equation is y = 0.6403 - 0.0016 x. This indicates that when the furazolidone concentration is 1~300 μM, the down-conversion fluorescence intensity of the amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 is linearly related to the furazolidone concentration, that is, after the present invention mixes and incubates furazolidone with a concentration gradient of 1~300 μM and the amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4, a linear relationship between the down-conversion fluorescence intensity and the furazolidone concentration can be established; then, after the furazolidone to be detected is mixed and incubated with it, the down-conversion emission intensity of the amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 is substituted into the linear relationship between the down-conversion fluorescence intensity and the concentration of furazolidone to obtain the concentration of the furazolidone to be detected.

[0101] Furthermore, in order to improve the sensitivity and accuracy of the test results, the Figure 4 and Figure 5 As can be seen from Figures c~d in the figure, a 980 nm near-infrared semiconductor laser or a 250 nm excitation light from a xenon lamp can be selected as the excitation light source according to the furazolidone concentration. Specifically, when the furazolidone concentration is between 0.05 and 30 μM, upconversion fluorescence detection with a 980 nm near-infrared semiconductor laser as the excitation light source can be selected. When the furazolidone concentration is between 1 and 300 μM, fluorescence detection with a 980 nm near-infrared semiconductor laser or a 250 nm light from a xenon lamp can be selected as the excitation light source.

[0102] Experimental Example 3

[0103] Experimental Example 3 of the present application performs a performance test on the amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 provided in Example 1.

[0104] Among them, the instruments used for performance testing include the Ocean Optics USB-2000+ fluorescence spectrometer, which uses a 980 nm infrared semiconductor laser as the excitation light source; and the American HORIBA Jobin Yvon fluorescence spectrometer, which uses a 250 nm xenon lamp as the excitation light source.

[0105] The performance test process is as follows: 300 μM of erythromycin, streptomycin, tobramycin, florfenicol, thiamphenicol, amoxicillin and furazolidone and other antibiotics were incubated with amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 at room temperature for 25 min, and then the up- and down-conversion fluorescence spectra were tested. The relative intensities of the up- and down-conversion fluorescence were as follows: Figure 6 As shown in Figures a~b in the figure, it can be seen from Figures a~b that after the addition of furazolidone, the up- and down-conversion fluorescence intensity of amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 can be quenched, indicating that amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 has good selectivity.

[0106] The performance test process also includes: mixing 300 μM erythromycin, streptomycin, tobramycin, florfenicol, thiamphenicol, and amoxicillin with 300 μM furazolidone respectively, and then incubating them with amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4 at room temperature for 25 minutes to test its up- and down-conversion fluorescence spectra. The up- and down-conversion fluorescence spectra are shown in FIG. Figure 7 As shown in Figures a~b in the figure, it can be seen from Figures a~b that there is no obvious change in the up-conversion and down-conversion fluorescence intensities, indicating that antibiotics such as erythromycin, streptomycin, tobramycin, florfenicol, thiamphenicol, and amoxicillin do not cause obvious interference in the detection of furazolidone by amino-silica-modified NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce@NaYF4. Based on this result, it is shown that amino-silica-modified NaYbF4:Tm / Gd@NaYF4:Tb@NaGdF4:Ce@NaYF4 has good anti-interference ability.

[0107] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A dual-mode fluorescent nanoprobe, characterized in that: The dual-mode fluorescent nanoprobe includes a core nanoparticle NaGdF4:Yb / Tm, a NaYF4:Tb inner shell, a NaGdF4:Ce middle shell, and a NaYF4 outer shell; The NaYF4:Tb inner shell layer covers the core nanoparticle NaGdF4:Yb / Tm; The NaGdF4:Ce intermediate shell layer covers the NaYF4:Tb inner shell layer; The NaYF4 outer shell layer covers the NaGdF4:Ce intermediate shell layer; In the core nanoparticles NaGdF4:Yb / Tm, the molar ratio of Yb: Tm: Gd is 40-80:0.5-2:20-60; In the NaYF4:Tb inner shell, the molar ratio of Y:Tb is 70-95:5-30; In the NaGdF4:Ce intermediate shell layer, the molar ratio of Gd: Ce is 70-90:10-20.

2. A dual-mode fluorescent nanoprobe according to claim 1, characterized in that: The particle size of the dual-mode fluorescent nanoprobe is 40-80 nm.

3. The dual-mode fluorescent nanoprobe according to claim 1, characterized in that: In the dual-mode fluorescent nanoprobe, the NaYF4 outer shell is modified with amino-silica.

4. The method for preparing a dual-mode fluorescent nanoprobe according to any one of claims 1 to 3, characterized in that: Step S1, adding ammonium fluoride and sodium hydroxide to a precursor reaction solution containing ytterbium / thulium / gadolinium-oleic acid complex to perform a core co-precipitation reaction to obtain core nanoparticles NaGdF4:Yb / Tm; Step S2: adding core nanoparticles NaGdF4:Yb / Tm, ammonium fluoride and sodium hydroxide to the yttrium-containing / terbium-oleic acid complex precursor reaction solution, the inner shell co-precipitation reaction was carried out to prepare the core-shell structure NaGdF4:Yb / Tm@NaYF4:Tb; Step S3, adding NaGdF4:Yb / Tm@NaYF4:Tb, ammonium fluoride and sodium hydroxide to the mixture containing gadolinium / The core-shell-shell structure of NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce was prepared by an intermediate shell co-precipitation reaction in the precursor reaction solution of the cerium-oleic acid complex. Step S4, NaGdF4:Yb / Tm@NaYF4:Tb@NaGdF4:Ce, ammonium fluoride, sodium hydroxide It is added into a precursor reaction solution containing yttrium-oleic acid complex to carry out outer shell co-precipitation reaction to prepare a dual-mode fluorescent nanoprobe with a core-shell-shell-shell structure.

5. The method for preparing a dual-mode fluorescent nanoprobe according to claim 4, characterized in that: After step S4, the method further includes the following steps: Step S5, dispersing the dual-mode fluorescent nanoprobe in an ethanol solvent, adding ethyl orthosilicate in an alkaline environment and hydrolyzing it to obtain a silica-modified dual-mode fluorescent nanoprobe; Step S6: adding 3-aminopropyltriethoxysilane to the silica-modified dual-mode fluorescent nanoprobe to perform an amination reaction to obtain the amination-silica-modified dual-mode fluorescent nanoprobe.

6. Use of a dual-mode fluorescent nanoprobe according to claim 3 in detecting antibiotics, characterized in that: The antibiotic is selected from furazolidone.

7. The use of a dual-mode fluorescent nanoprobe in detecting antibiotics according to claim 6, characterized in that: The application is specifically: application in the detection of antibiotic pollution in water bodies.

8. The use of a dual-mode fluorescent nanoprobe in detecting antibiotics according to claim 6, characterized in that: The application process includes the steps of: Step 1: mixing the dual-mode fluorescent nanoprobe with a concentration gradient antibiotic standard solution and performing fluorescence detection to establish a linear relationship between the concentration gradient antibiotic standard solution and the emission intensity of the dual-mode fluorescent nanoprobe; Step 2: mixing the dual-mode fluorescent nanoprobe and the antibiotic sample to be detected, and then performing fluorescence detection to obtain the emission intensity of the dual-mode fluorescent nanoprobe to be detected; Step 3: Substitute the emission intensity of the dual-mode fluorescent nanoprobe into the linear relationship between the concentration gradient of the antibiotic standard solution and the emission intensity of the dual-mode fluorescent nanoprobe to calculate the concentration of the antibiotic in the antibiotic sample to be detected.

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