An upconversion fluorescent nanoprobe, its preparation method, and its application

Abstract

This application belongs to the field of antibiotic detection technology, and particularly relates to an upconversion fluorescent nanoprobe, its preparation method, and its application. The hyaluronic acid (HYA) modified upconversion fluorescent nanoprobe NaGdF4:Yb / Tm@NaYF4:Eu@NaYF4 provided in this patent application exhibits significantly quenched fluorescence emission intensity when antibiotics such as nitrofurazone (NFZ), nitrofurantoin (NFT), and doxorubicin (DOX) are added. Furthermore, the fluorescence emission intensity of the upconversion fluorescent nanoprobe gradually decreases as the concentration of the added antibiotic increases. Thus, quantitative detection of multiple antibiotics can be achieved based on the correlation between antibiotic concentration and the change in upconversion emission intensity. This fluorescence detection method also boasts strong anti-interference capabilities, high sensitivity, low cost, and simple operation, solving the technical problem of the lack of fluorescent probes for antibiotic detection in the prior art.

Description

Technical Field

[0001] This application belongs to the field of antibiotic detection technology, and in particular relates to an upconversion fluorescent nanoprobe, its preparation method, and its application. Background Technology

[0002] Antibiotics play a crucial role in preventing the spread of harmful microorganisms and treating various diseases, and are widely used in animal husbandry, agriculture, aquaculture, and human health. However, most antibiotics are not fully utilized by humans and animals, resulting in the release of a large portion of them into the environment, polluting water and the food chain. They accumulate in the human body through contaminated food, affecting normal metabolism, disrupting the microbial ecosystem, and directly threatening human health. Therefore, it is necessary to test the residual levels of antibiotics in the aquatic environment.

[0003] Currently, methods for detecting antibiotic residues mainly include liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis (CES), and high-performance liquid chromatography (HPLC). However, these methods suffer from drawbacks such as complex sample pretreatment, difficult and time-consuming operation, the need for specialized personnel, the requirement for expensive and bulky instruments like LC-MS, CES, and HPLC, and relatively low sensitivity and selectivity. These drawbacks make the entire analytical process expensive and inefficient, limiting their practical application. Compared to LC-MS, CES, and HPLC, fluorescence detection uses a relatively inexpensive fluorescence spectrometer, has simpler sample pretreatment and detection operations, and can achieve both qualitative and quantitative detection of samples, making it a more suitable detection method for practical applications.

[0004] Fluorescence detection relies on the interaction between fluorescent molecules, such as fluorescent probes, and the sample to be tested, resulting in fluorescence emission under excitation light. The qualitative and quantitative detection of the sample is achieved based on the intensity of the emitted fluorescence. However, there is currently a lack of fluorescent probes that can detect antibiotics. Summary of the Invention

[0005] In view of this, this application provides an upconversion fluorescent nanoprobe, its preparation method, and its application, to solve the technical problem of the lack of fluorescent probes for detecting antibiotics in the prior art.

[0006] The first aspect of this application provides an upconversion fluorescent nanoprobe, which includes a core nanoparticle NaGdF4:Yb / Tm, an inner shell NaYF4:Eu, and an outer shell NaYF4.

[0007] The core nanoparticles NaGdF4:Yb / Tm are coated with an inner shell NaYF4:Eu;

[0008] The inner shell NaYF4:Eu is covered by the outer shell NaYF4.

[0009] Preferably, the upconversion fluorescent nanoprobe further includes hyaluronic acid;

[0010] The hyaluronic acid is coated onto the surface of the upconversion fluorescent nanoprobe via electrostatic interaction.

[0011] Preferably, the particle size of the upconversion fluorescent nanoprobe is 20–80 nm.

[0012] Preferably, in NaGdF4:Yb / Tm, the molar ratio of Gd:Yb:Tm is 47–69.5 mol%: 30–50 mol%: 0.5–3 mol%.

[0013] In the NaYF4:Eu mixture, Y:Eu = 80–95 mol% : 5–20 mol%.

[0014] The second aspect of this application provides a method for preparing upconversion fluorescent nanoprobes, which can prepare the upconversion fluorescent nanoprobes described in the first aspect. The preparation method includes the following steps:

[0015] Step S1: Ammonium fluoride and sodium hydroxide are added to the precursor reaction solution containing gadolinium / ytterbium / thulium-oleic acid complex to carry out a core coprecipitation reaction to prepare core nanoparticles NaGdF4:Yb / Tm;

[0016] Step S2: Add the core nanoparticles NaGdF4:Yb / Tm, ammonium fluoride, and sodium hydroxide to the precursor reaction solution containing yttrium / europium-oleic acid complex to carry out an inner-shell co-precipitation reaction, and prepare the core-shell structured NaGdF4:Yb / Tm@NaYF4:Eu.

[0017] Step S3: Add NaGdF4:Yb / Tm@NaYF4:Eu, ammonium fluoride, and sodium hydroxide to the precursor reaction solution containing yttrium-oleic acid complex to carry out an outer shell co-precipitation reaction, thereby preparing a core-shell-shell structured NaGdF4:Yb / Tm@NaYF4:Eu@NaYF4.

[0018] Preferably, after step S3, the following step is also included:

[0019] Step S4: Remove the oleic acid from the surface of NaGdF4:Yb / Tm@NaYF4:Eu@NaYF4 to obtain oleic acid-free NaGdF4:Yb / Tm@NaYF4:Eu@NaYF4;

[0020] Step S5: Stir NaGdF4:Yb / Tm@NaYF4:Eu@NaYF4 with oleic acid removed and hyaluronic acid to obtain upconversion fluorescent nanoprobes.

[0021] Preferably, in step S1, the method for preparing the precursor reaction solution of the gadolinium / ytterbium / thulium-oleic acid complex includes: performing a coordination reaction of gadolinium salt, ytterbium salt, thulium salt, oleic acid and 1-octadecene to prepare the precursor reaction solution of the gadolinium / ytterbium / thulium-oleic acid complex;

[0022] In step S2, the preparation method of the precursor reaction solution of the yttrium / europium-oleic acid complex includes: performing a coordination reaction of yttrium salt, europium salt, oleic acid and 1-octadecene to prepare the precursor reaction solution of the yttrium / europium-oleic acid complex;

[0023] In step S3, the preparation method of the precursor reaction solution of the yttrium-oleic acid complex includes: carrying out a coordination reaction of yttrium salt, oleic acid and 1-octadecene to prepare the precursor reaction solution of the yttrium-oleic acid complex.

[0024] Preferably, in steps S1 to S3, the temperature of the coordination reaction is 150-180°C and the time is 20-40 min.

[0025] Preferably, in step S1, the process of the nuclear coprecipitation reaction is as follows: heating to 50°C and reacting for 30 min, heating to 110°C and reacting for 25 min, removing methanol, evacuating for 10 min and then heating to 300°C, and reacting for 1 h in an argon atmosphere.

[0026] In step S2, the co-precipitation reaction of the inner shell is as follows: the temperature is raised to 50°C and reacted for 30 min, the temperature is raised to 110°C and reacted for 25 min, methanol is removed, vacuum is applied for 10 min, and then the temperature is raised to 300°C and reacted for 1 h in an argon atmosphere.

[0027] In step S3, the outer shell coprecipitation reaction process is as follows: heat to 50°C and react for 30 min, heat to 110°C and react for 25 min, remove methanol, evacuate for 10 min, heat to 300°C, and react for 1 h in an argon atmosphere.

[0028] Preferably, in step S4, the preparation method of the oleic acid-removed NaGdF4:Yb / Tm@NaYF4:Eu@NaYF4 includes: dispersing NaGdF4:Yb / Tm@NaYF4:Eu@NaYF4 in hydrochloric acid aqueous solution, and performing ultrasonic, centrifugation and washing steps sequentially at room temperature to prepare oleic acid-removed NaGdF4:Yb / Tm@NaYF4:Eu@NaYF4.

[0029] Preferably, in step S5, the stirring time is 10 to 180 minutes.

[0030] The third aspect of this application provides the application of the upconversion fluorescent nanoprobes described in the first or second aspect in antibiotic detection.

[0031] Preferably, the application is specifically in the detection of antibiotic pollution in water bodies.

[0032] Preferably, the antibiotics include: nitrofurazone, nitrofurantoin, and doxorubicin.

[0033] Preferably, the application process includes the following steps:

[0034] Step 1: Mix the upconversion fluorescent nanoprobe with antibiotic standard solutions of varying concentrations and perform fluorescence detection to establish a linear relationship between the emission intensity of the antibiotic standard solutions of varying concentrations and the upconversion fluorescent nanoprobe.

[0035] Step 2: Mix the upconversion fluorescent nanoprobe with the antibiotic sample to be tested and then perform fluorescence detection to obtain the emission intensity of the upconversion fluorescent nanoprobe to be tested;

[0036] Step 3: Substitute the emission intensity of the upconversion fluorescent nanoprobe to be detected into the linear relationship between the concentration gradient of the antibiotic standard solution and the emission intensity of the upconversion fluorescent nanoprobe, and calculate the concentration of antibiotic in the antibiotic sample to be detected.

[0037] In summary, this application provides an upconversion fluorescent nanoprobe, its preparation method, and its application. The provided upconversion fluorescent nanoprobe is a NaGdF4:Yb / Tm nanoparticle sequentially coated with an inner shell of NaYF4:Eu and an outer shell of NaYF4. This upconversion fluorescent nanoprobe is a lanthanide ion-doped upconversion nanoparticle capable of converting low-energy, long-wavelength near-infrared (NIR) excitation light into higher-energy, shorter-wavelength ultraviolet (UV) or visible light emission light. Upon addition of antibiotics such as nitrofurazone (NFZ), nitrofurantoin (NFT), and doxorubicin (DOX), the fluorescence emission intensity of the upconversion fluorescent nanoprobe is significantly quenched. Furthermore, as the concentration of the added antibiotic increases, the fluorescence emission intensity of the upconversion fluorescent nanoprobe gradually decreases. Therefore, based on the correlation between antibiotic concentration and the change in upconversion emission intensity, quantitative detection of multiple antibiotics can be achieved, thereby solving the technical problem of the lack of fluorescent probes for antibiotic detection in the prior art. Attached Figure Description

[0038] 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.

[0039] Figure 1The characterization results of the upconversion fluorescent nanoprobes prepared in Example 2 of this application are as follows: (a) is a TEM image of the oleic acid-coated upconversion fluorescent nanoprobes; (b) is a TEM image of the upconversion fluorescent nanoprobes selected for compositional analysis; (cg) are the elemental distribution diagrams of Gd, Yb, Tm, Y, and Eu in the upconversion fluorescent nanoprobes; (h) is the EDS elemental analysis of the upconversion fluorescent nanoprobes; and (i) is the HRTEM image of a single upconversion fluorescent nanoprobe.

[0040] Figure 2 The following are characterization results of the surface modification of the upconversion fluorescent nanoprobes prepared in Example 2 of this application: (a) TEM image of the ligand-free upconversion fluorescent nanoprobe; (b) TEM image of the hyaluronic acid-modified upconversion fluorescent nanoprobe; (c) FT-IR spectra of the oleic acid-coated upconversion fluorescent nanoprobe, the oleic acid-free upconversion fluorescent nanoprobe, hyaluronic acid, and the hyaluronic acid-modified upconversion fluorescent nanoprobe; (d) Zeta potential of the oleic acid-free upconversion fluorescent nanoprobe and the hyaluronic acid-modified upconversion fluorescent nanoprobe; (e) Upconversion fluorescence spectra of the hyaluronic acid-modified upconversion fluorescent nanoprobe, the oleic acid-free upconversion fluorescent nanoprobe, and the oleic acid-coated upconversion fluorescent nanoprobe under 980 nm laser excitation; (f) Upconversion fluorescence spectra of the hyaluronic acid-modified upconversion fluorescent nanoprobe after the addition of nitrofurazone (NFZ), nitrofurantoin (NFT), and doxorubicin (DOX), respectively.

[0041] Figure 3 The UV-Vis absorption spectra of the antibiotics in aqueous solution in Experiment Example 1 are shown in (a) and (b) are UV-Vis absorption spectra of different antibiotics (~5 μM) in aqueous solution.

[0042] Figure 4 The images show the upconversion fluorescence spectrum of UCNPs-HYA after the addition of NFZ in Experiment Example 2, as well as the linear relationship between different concentrations of NFZ and the upconversion emission intensity. (a) shows the upconversion fluorescence spectrum of UCNPs-HYA after the addition of NFZ under 980 nm laser excitation, 4 W power, and 400 ms integration time. (b) shows the upconversion emission intensity ratio (Ig). 360 / I 615 (c) shows the response of NFZ concentrations (0–200 μM), and (d) shows the upconversion emission intensity ratio (I). 360 / I 615 The linear response of NFZ concentration (2.5–20 μM) to 1 / 2 NFZ concentration is shown in the graph, and (d) represents the upconversion emission intensity ratio (I). 360 / I 615Linear response graph of NFZ concentration (30-100 μM);

[0043] Figure 5 The images show the upconversion fluorescence spectrum of UCNPs-HYA after the addition of NFT in Experiment Example 2, and the linear relationship between different concentrations of NFT and upconversion emission intensity. (a) shows the upconversion fluorescence spectrum of UCNPs-HYA after the addition of NFT under 980 nm laser excitation, 4 W power, and 400 ms integration time. (b) shows the upconversion emission intensity ratio (Ig). 360 / I 615 (c) shows the response of NFT concentrations (0–100 μM) to NFT concentrations. 360 / I 615 Linear response relationship of NFT concentration (2.5–20 μM), (d) upconversion emission intensity ratio (I 360 / I 615 Linear response plot of NFT concentration (30–80 μM);

[0044] Figure 6 The images show the upconversion fluorescence spectrum of UCNPs-HYA after the addition of DOX in Experiment Example 2, as well as the linear relationship between different concentrations of DOX and the upconversion emission intensity. (a) shows the upconversion fluorescence spectrum of UCNPs-HYA after the addition of DOX under 980 nm laser excitation, 4 W power, and 400 ms integration time. (b) shows the upconversion emission intensity ratio (Ig). 475 / I 615 (c) Relationship between DOX concentration (0-200 μM) and upconversion emission intensity ratio (I) 475 / I 615 Linear response to DOX concentrations (2.5–60 μM), (d) upconversion emission intensity ratio (I 475 / I 615 Linear response relationship of DOX concentration (80-200 μM) to DOX concentration.

[0045] Figure 7 The relative upconversion emission intensity (IL) of UCNPs-HYA after adding different concentrations of antibiotic substances in Experiment Example 3. 360 / I 615 ) and (I 475 / I 615 )picture. Detailed Implementation

[0046] This application provides an upconversion fluorescent nanoprobe, its preparation method, and its application, which addresses the technical problem of the lack of fluorescent probes for detecting antibiotics in the prior art.

[0047] 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.

[0048] In this embodiment, the upconversion fluorescent nanoprobe is UCNPs;

[0049] Oleic acid-coated upconversion fluorescent nanoprobes are OA-UCNPs;

[0050] Hyaluronic acid is HYA;

[0051] The hyaluronic acid-modified upconversion fluorescent nanoprobe is UCNPs-HYA;

[0052] Among antibiotics, nitrofurazone is NFZ, nitrofurantoin is NFT, and doxorubicin is DOX.

[0053] The instruments used included: a Japanese HT7700 transmission electron microscope with an operating voltage of 100kV; and a US Thermo-Filsher-Nicolet 6700 microscope with a scanning range of 4000–500 cm⁻¹. -1 The resolution is 2cm. -1 The instrument was used for 32 scans; a PerkinElmer Lambda 950 UV-Vis-NIR spectrophotometer was used, with a scanning wavelength range of 200–800 nm; a Zeta potential analysis was performed using a Zetasizer NANOZS nanoparticle size and Zeta potential analyzer; and a Marine Optics USB2000+ fluorescence spectrometer was used, with a 980 nm infrared semiconductor laser as the excitation source.

[0054] Example 1

[0055] Given the current lack of fluorescent probes for detecting antibiotics, this application provides an upconversion fluorescent nanoprobe with a core-shell-shell structure. The nanoprobe comprises a core nanoparticle NaGdF4:Yb / Tm, an inner shell NaYF4:Eu, and an outer shell NaYF4. The structure consists of the core nanoparticle NaGdF4:Yb / Tm coated with the inner shell NaYF4:Eu, and the inner shell NaYF4:Eu coated with the outer shell NaYF4. This upconversion fluorescent nanoprobe is a lanthanide-doped upconversion nanoparticle capable of converting low-energy, long-wavelength near-infrared (NIR) excitation light into higher-energy, shorter-wavelength ultraviolet (UV) or visible light emission. Upon addition of antibiotics such as nitrofurazone (NF2), nitrofurantoin (NFT), and doxorubicin (DOX), the fluorescence of the upconversion fluorescent nanoprobe increases. The emission intensity is significantly quenched, and the fluorescence emission intensity of the upconversion fluorescent nanoprobe gradually decreases with increasing antibiotic concentration. This allows for the quantitative detection of multiple antibiotics based on the correlation between antibiotic concentration and the change in upconversion emission intensity. Simultaneously, the upconversion red emission intensity remains constant, serving as an internal reference for fluorescence intensity in upconversion fluorescent nanoprobe detection. Furthermore, low-energy near-infrared excitation avoids photodamage, offers greater penetration depth into biological tissues, eliminates background fluorescence interference, and provides a higher signal-to-noise ratio, facilitating the construction of a highly sensitive sensing system and improving detection sensitivity. Therefore, the upconversion fluorescent nanoprobe provided in this application not only overcomes the current lack of fluorescent probes for antibiotic detection but also represents a detection method with strong anti-interference capabilities, high sensitivity, and excellent selectivity for antibiotics.

[0056] As a preferred option, since most antibiotics are found in water, the dispersibility of upconversion fluorescent nanoprobes in water significantly affects their detection performance. Therefore, this application modifies the upconversion fluorescent nanoprobes by using the electrostatic interaction of positive and negative charges to modify the surface of the upconversion fluorescent nanoprobes with hyaluronic acid.

[0057] Preferably, for the size of the upconversion fluorescent nanoprobe, this application prefers upconversion fluorescent nanoprobes with a particle size of 20-80 nm.

[0058] Preferably, for the proportions of each component in the upconversion fluorescent nanoprobe, in NaGdF4:Yb / Tm@NaYF4:Eu@NaYF4, the molar ratio of Gd:Yb:Tm in NaGdF4:Yb / Tm is 50mol%:49mol%:1mol%; the ratio of Y:Eu in NaYF4:Eu is 90mol%:10mol%; and the ratio of NaGdF4:Yb / Tm@NaYF4:Eu@NaYF4 to hyaluronic acid in the upconversion fluorescent nanoprobe is 1mol:0.5mol.

[0059] Example 2

[0060] Example 2 of this application provides a method for preparing the upconversion fluorescent nanoprobe described in Example 1. The preparation method synthesizes an upconversion fluorescent nanoprobe NaGdF4:Yb / Tm(49 / 1mol%)@NaYF4:Eu(10mol%)@NaYF4 with a particle size of approximately 60nm. The preparation method includes preparing a precursor reaction solution of a rare earth-oleic acid complex, performing a coprecipitation reaction, preparing a cyclohexane solution, removing oleic acid, and modifying hyaluronic acid.

[0061] The process of preparing the precursor reaction solution of rare earth-oleic acid complex includes: adding gadolinium acetate (0.2 mmol), ytterbium acetate (0.196 mmol), thulium acetate (0.004 mmol), oleic acid (4 mL) and 1-octadecane (6 mL) into a two-necked flask; heating to 160 °C in a heating mantle for 30 min; removing water from the reaction system; and cooling to room temperature to obtain the precursor reaction solution containing gadolinium / ytterbium / thulium-oleic acid complex.

[0062] Yttrium acetate (0.36 mmol), europium acetate (0.04 mmol), oleic acid (4 mL) and 1-octadecane (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 a precursor reaction solution containing the yttrium / europium-oleic acid complex.

[0063] Yttrium acetate (0.4 mmol), oleic acid (4 mL), and 1-octadecene (6 mL) were added to a two-necked flask; the mixture was heated to 160 °C for 30 min in a heating mantle, the water in the reaction system was removed, and the mixture was cooled to room temperature to obtain a precursor reaction solution containing the yttrium / -oleic acid complex.

[0064] The coprecipitation reaction process includes: adding NH4F (1.52 mmol) and NaOH (1 mmol) to the precursor reaction solution containing gadolinium / ytterbium / thulium-oleic acid complex, heating the mixture to 50 °C and reacting for 30 min; heating to 110 °C and reacting for 25 min to remove methanol; evacuating for 10 min and then heating to 300 °C and reacting for 1 h under an argon atmosphere to obtain oleic acid-coated core nanoparticles with the composition NaGdF4:Yb / Tm (Gd:Yb:Tm=50mol%:49mol%:1mol%).

[0065] The process of preparing the cyclohexane solution includes: cooling to room temperature after the reaction is completed, transferring the oleic acid-coated core nanoparticle reaction solution to a centrifuge tube, adding anhydrous ethanol (4 mL, 99.5%), shaking to mix, centrifuging at 6000 rpm for 3 min, and removing the supernatant; adding cyclohexane (4 mL, 99.5%), adding anhydrous ethanol (8 mL, 99.5%), shaking to mix, centrifuging at 6000 rpm for 3 min, and removing the supernatant; adding cyclohexane (4 mL, 99.5%), anhydrous ethanol (4 mL, 99.5%), and methanol (4 mL, 99.5%), shaking to mix, centrifuging at 6000 rpm for 3 min, and removing the supernatant to obtain oleic acid-coated core nanoparticles; dispersing them in cyclohexane (6.5 mL, 99.5%), sealing and storing at low temperature to obtain an oleic acid-coated core nanoparticle NaGdF4:Yb / Tm cyclohexane dispersion.

[0066] The coprecipitation reaction process includes: adding 3 mL of NaGdF4:Yb / Tm cyclohexane dispersion of core nanoparticles, 1.52 mmol of NH4F, and 1 mmol of NaOH to a reaction solution containing a rare earth-oleic acid complex precursor; heating the mixture to 50 °C and reacting for 30 min; heating to 110 °C and reacting for 25 min to remove methanol; evacuating for 10 min and then heating to 300 °C and reacting for 1 h under an argon atmosphere to obtain oleic acid-coated core-shell structured nanoparticles with the composition NaGdF4:Yb / Tm (Gd:Yb:Tm = 50 mol%: 49 mol%: 1 mol%) @ NaYF4:Eu (Y:Eu = 90 mol%: 10 mol%).

[0067] The process of preparing the cyclohexane solution includes: transferring the reaction solution of oleic acid-coated core-shell nanoparticles NaGdF4:Yb / Tm@NaYF4:Eu to a centrifuge tube, adding anhydrous ethanol (4 mL, 99.5%), shaking to mix, centrifuging at 6000 rpm for 3 min, and removing the supernatant; adding cyclohexane (4 mL, 99.5%), anhydrous ethanol (8 mL, 99.5%), shaking to mix, centrifuging at 6000 rpm for 3 min, and removing the supernatant; adding cyclohexane (4 mL, 99.5%), anhydrous ethanol (4 mL, 99.5%), and methanol (4 mL, 99.5%), shaking to mix, centrifuging at 6000 rpm for 3 min, and removing the supernatant to obtain oleic acid-coated core-shell nanoparticles; dispersing them in cyclohexane (4 mL, 99.5%) to prepare a NaGdF4:Yb / Tm@NaYF4:Eu cyclohexane dispersion.

[0068] The coprecipitation reaction process includes: adding 3.9 mL of NaGdF4:Yb / Tm@NaYF4:Eu cyclohexane dispersion, 1.52 mmol of NH4F, and 1 mmol of NaOH to the precursor reaction solution containing the rare earth-oleic acid complex; heating the mixture to 50 °C and reacting for 30 min; heating to 110 °C and reacting for 25 min to remove methanol; evacuating for 10 min and then heating to 300 °C and reacting for 1 h under an argon atmosphere to obtain oleic acid-coated core-shell-shell structured rare earth-doped UCNPs with the composition NaGdF4:Yb / Tm (Gd:Yb:Tm = 50 mol%: 49 mol%: 1 mol%)@NaYF4:Eu (Y:Eu = 90 mol%: 10 mol%)@NaYF4 (Y = 100 mol%), abbreviated as core-shell-shell structured UCNPs-OA.

[0069] The process of preparing the cyclohexane solution includes: transferring the UCNPs-OA reaction solution to a centrifuge tube, adding anhydrous ethanol (4 mL, 99.5%), shaking to mix evenly, centrifuging at 6000 rpm for 3 min, and removing the supernatant; adding cyclohexane (4 mL, 99.5%), anhydrous ethanol (8 mL, 99.5%), shaking to mix evenly, centrifuging at 6000 rpm for 3 min, and removing the supernatant; adding cyclohexane (4 mL, 99.5%), anhydrous ethanol (4 mL, 99.5%), and methanol (4 mL, 99.5%), shaking to mix evenly, centrifuging at 6000 rpm for 3 min, and removing the supernatant; dispersing the obtained UCNPs-OA in cyclohexane (3.9 mL, 99.5%) to prepare the OA-UCNPs cyclohexane dispersion.

[0070] The process for removing oleic acid includes: adding 3.8 mL of anhydrous ethanol (3 mL, 99.5%) to a cyclohexane dispersion of OA-UCNPs, shaking until homogeneous, and centrifuging at 6000 rpm for 1 min to precipitate OA-UCNPs from cyclohexane, and removing the supernatant; dispersing the obtained OA-UCNPs in hydrochloric acid aqueous solution (3.8 mL, 0.1 M), sonicating at 28 °C for 1 h, centrifuging at 15000 rpm for 15 min, and collecting the obtained particles; washing 2-3 times with ultrapure water, dispersing the particles in 3.8 mL of ultrapure water, sealing and storing at low temperature to obtain UCNPs with oleic acid ligands removed.

[0071] The process of modifying hyaluronic acid included: adding 0.1 mL of UCNPs (10 mg / mL) with oleic acid ligands removed to 0.05 mL of HYA (1 mg / mL) and stirring at room temperature. The polyanionic HYA, containing functional groups such as carboxyl and hydroxyl groups, was electrostatically coated onto the positively charged UCNPs surface with oleic acid ligands removed; the stirring time was 20 min, resulting in a UCNPs-HYA probe solution with a concentration of 0.5 mg / mL.

[0072] Experimental Example 1

[0073] Experimental Example 1 of this application tests the performance of the upconversion fluorescent nanoprobe prepared in Example 2. The test results are as follows: Figure 1-7 As shown.

[0074] from Figure 1 It can be seen that the average diameter of well-dispersed OA-UCNPs is 65.6 nm; Gd 3+ Yb 3+ and Tm 3+ Distributed in nuclear particles, Eu 3+ Distributed in the inner shell, Y 3+ The UCNPs are distributed in both the inner and outer shells. The lattice fringe spacing of the UCNPs is 0.504 nm, which corresponds to the (100) plane of the hexagonal phase structure, indicating that the prepared UCNPs have a good crystal structure.

[0075] from Figure 2 It can be seen that ligand-free UCNPs are uniform in size with a particle diameter of 65.3 nm, while UCNPs-HYA are spherical particles with uniform size and an average diameter of 64.6 nm; UCNPs-HYA at 3438 cm⁻¹... -1 There is a broad peak at 2925 cm⁻¹ related to the stretching vibration of the -OH group. -1 and 2850cm -1 The double peak at 1649 cm⁻¹ corresponds to the asymmetric and symmetric stretching vibrations of the CH₂ group. -1 The peak is attributed to the carboxyl group, at 1568 cm⁻¹. -1 and 1415cm -1 The peak is due to the bending vibration of NH and the stretching vibration of CO and OCO groups, 1021 cm⁻¹. -1The peak is due to the CO symmetric stretching vibration, indicating that the UCNPs surface was successfully coated with HYA. Simultaneously, the ligandless UCNPs are positively charged due to surface protonation, with a potential of 31±2 mV, allowing them to further bind to negatively charged functional groups. The UCNPs-HYA surface is negatively charged, with a potential of -8±0.2 mV, due to the -COOH functional group from the polyanionic HYA. The ligandless UCNPs and UCNPs-HYA exhibit upconversion fluorescence intensities similar to OA-UCNPs, indicating that ligand removal and HYA surface modification have minimal impact on the upconversion fluorescence performance of UCNPs. This demonstrates that the hyaluronic acid-modified upconversion fluorescent nanoprobe UCNPs-HYA can improve the water solubility and dispersibility of UCNPs without affecting their fluorescence properties.

[0076] At the same time, it can also be seen from Figure 2 It can be seen that after adding NFZ / NFT, the upconversion emission intensity at 344nm and 360nm of UCNPs-HYA decreased significantly; after adding DOX, the upconversion emission at 449nm and 476nm of UCNPs-HYA was effectively quenched, while the upconversion red light emission intensity at 615nm of UCNPs-HYA remained basically unchanged, which can be used as an internal standard for fluorescence detection. This indicates that the fluorescent probe UCNPs-HYA can be used to detect the concentration of NFZ / NFT / DOX.

[0077] from Figure 3 It can be seen that NFZ and NFT have broad absorption in the range of 200–460 nm, while DOX has broad absorption in the range of 200–570 nm. Figure 3 (b) As shown in the UV-Vis absorption spectrum, UCNPs-HYA does not have obvious absorption peaks, but after the addition of NFZ, NFT and DOX, the UV-Vis spectrum shows the characteristic absorption of the above substances.

[0078] Experiment Example 2

[0079] Experimental Example 2 of this application demonstrates the detection of NFZ / NFT / DOX using an upconversion fluorescent nanoprobe.

[0080] The detection of NFZ includes adding different concentrations of NFZ, incubating at room temperature for 20 minutes, and using the change in upconversion fluorescence intensity to detect the concentration of the target substance NFZ.

[0081] The results are as follows Figure 4As shown, with the gradual increase of NFZ concentration (0–200 μM), the emission intensity at 344 nm and 360 nm of the upconversion emission of UCNPs-HYA gradually decreases. Furthermore, when the NFZ concentration is in the range of 2.5–20 μM, the relative upconversion emission intensity of UCNPs-HYA exhibits a good linear relationship with the NFZ concentration, with the linear equation y = 1.198 - 0.0233x. Conversely, when the NFZ concentration is in the range of 30–100 μM, the relative upconversion emission intensity of UCNPs-HYA also exhibits a good linear relationship with the NFZ concentration, with the linear equation y = 0.8196 - 0.0067x; This indicates that when the NFZ concentration is 2.5–100 μM, the upconversion fluorescence intensity of the UCNPs-HYA probe is linearly related to the NFZ concentration. That is, after mixing and incubating NFZ and upconversion fluorescent nanoprobes with a concentration gradient of 2.5–100 μM, a linear relationship between the upconversion fluorescence intensity and the NFZ concentration can be established. Then, after mixing and incubating the NFZ to be detected and the fluorescent nanoprobes, the concentration of the NFZ to be detected can be obtained by substituting the emission intensity of the obtained upconversion fluorescent nanoprobes into the linear relationship between the upconversion fluorescence intensity and the NFZ concentration.

[0082] The detection of NFTs involves adding different concentrations of NFTs, incubating at room temperature for 20 minutes, and then using the change in upconversion fluorescence intensity to detect the concentration of the target NFT.

[0083] The results are as follows Figure 5 As shown, with the gradual increase of NFT concentration (0–100 μM), the emission intensity at 344 nm and 360 nm of the upconversion emission of UCNPs-HYA gradually decreases. Furthermore, when the NFT concentration is in the range of 2.5–20 μM, the relative upconversion emission intensity of UCNPs-HYA exhibits a good linear relationship with the NFT concentration, with the regression linear equation being y = 1.319 - 0.0315x. When the NFT concentration is in the range of 30–80 μM, the relative upconversion emission intensity of UCNPs-HYA also exhibits a good linear relationship with the NFT concentration, with the linear equation being y = 0.7501. -0.0082x; This indicates that the upconversion fluorescence intensity of the UCNPs-HYA probe is linearly related to the NFT concentration when the NFT concentration is 2.5–80 μM. That is, after mixing and incubating NFT and upconversion fluorescent nanoprobes with a concentration gradient of 2.5–80 μM, a linear relationship between the upconversion fluorescence intensity and the NFT concentration can be established. Then, after mixing and incubating the NFT to be detected and the fluorescent nanoprobes, the concentration of the NFT to be detected can be obtained by substituting the emission intensity of the obtained upconversion fluorescent nanoprobes into the linear relationship between the upconversion fluorescence intensity and the NFT concentration.

[0084] DOX detection: Different concentrations of DOX were added and incubated at room temperature for 20 min. The concentration of the target substance NFT was detected by the change in upconversion fluorescence intensity.

[0085] The results are as follows Figure 6 As shown, with the gradual increase of DOX concentration (0–200 μM), the emission intensity at 450 nm and 476 nm of the upconversion emission of UCNPs-HYA gradually decreases. Furthermore, when the DOX concentration is in the range of 2.5–60 μM, the relative upconversion emission intensity of UCNPs-HYA exhibits a good linear relationship with the DOX concentration, with the regression linear equation being y = 4.634 - 0.0381x. When the DOX concentration is in the range of 80–200 μM, the relative upconversion emission intensity of UCNPs-HYA also exhibits a good linear relationship with the DOX concentration, with the linear equation being y = 2.793 - 0.0106x; This indicates that when the DOX concentration is 2.5–200 μM, the upconversion fluorescence intensity of the UCNPs-HYA probe is linearly related to the DOX concentration. That is, after mixing and incubating DOX and upconversion fluorescent nanoprobes with a concentration gradient of 2.5–200 μM, a linear relationship between the upconversion fluorescence intensity and the DOX concentration can be established. Then, after mixing and incubating the DOX to be detected and the fluorescent nanoprobes, the concentration of the DOX to be detected can be obtained by substituting the emission intensity of the obtained upconversion fluorescent nanoprobes into the linear relationship between the upconversion fluorescence intensity and the DOX concentration.

[0086] Experimental Example 3

[0087] Experimental Example 3 of this application demonstrates the detection of multiple antibiotics using upconversion fluorescent nanoprobes.

[0088] The detection process included incubating different antibiotic substances (ERY, AMX, SMZ, FFC, CAP, ONZ, THP, NFZ, NFT, and DOX) at equal concentrations (200 μM) with upconversion fluorescent nanoprobes at room temperature for 20 min, and then measuring the upconversion fluorescence spectra.

[0089] The results are as follows Figure 7 As shown, apart from the negligible fluorescence quenching after the addition of ONZ, only the addition of NFZ, NFT and DOX resulted in significant quenching of the upconversion fluorescence intensity. The upconversion fluorescence intensity ratio did not change significantly after the addition of other substances, indicating that the fluorescent probe has good selectivity and can be used for the detection of specific antibiotics.

[0090] From Experiment 1-2 and Figures 1-7As can be seen, the fluorescence emission intensity of the upconversion fluorescent nanoprobe provided in this application is significantly quenched after the addition of antibiotics such as nitrofurazone (NFZ), nitrofurantoin (NFT), and doxorubicin (DOX). Furthermore, the fluorescence emission intensity gradually decreases with increasing antibiotic concentration. This allows for the quantitative detection of multiple antibiotics based on the correlation between antibiotic concentration and the change in upconversion emission intensity. Simultaneously, the upconversion red light emission intensity remains constant, serving as an internal reference for fluorescence detection using the upconversion fluorescent nanoprobe. Moreover, low-energy near-infrared excitation avoids photodamage, provides greater penetration depth into biological tissues, eliminates background fluorescence interference, and offers a higher signal-to-noise ratio, facilitating the construction of a highly sensitive sensing system and improving detection sensitivity. Therefore, the upconversion fluorescent nanoprobe provided in this application not only overcomes the current lack of fluorescent probes for antibiotic detection but also represents a detection method with strong anti-interference capabilities, high sensitivity, and excellent selectivity for antibiotics.

[0091] 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. An application of an upconversion fluorescent nanoprobe in antibiotic detection, characterized in that, The upconversion fluorescent nanoprobe comprises a core nanoparticle NaGdF4:Yb / Tm, an inner shell NaYF4:Eu, and an outer shell NaYF4. The core nanoparticles NaGdF4:Yb / Tm are coated with the inner shell NaYF4:Eu; The inner shell NaYF4:Eu is covered by the outer shell NaYF4; The antibiotics include: nitrofurazone, nitrofurantoin, and doxorubicin; The upconversion fluorescent nanoprobe also includes hyaluronic acid; The hyaluronic acid is coated onto the surface of the upconversion fluorescent nanoprobe via electrostatic interaction.

2. The application of the upconversion fluorescent nanoprobe according to claim 1 in antibiotic detection, characterized in that, The upconversion fluorescent nanoprobe has a particle size of 20~80 nm.

3. The application of the upconversion fluorescent nanoprobe according to claim 1 in antibiotic detection, characterized in that, In the NaGdF4:Yb / Tm, the molar ratio of Gd:Yb:Tm is 47~69.5 mol%:30~50 mol%:0.5~3 mol%; In the NaYF4:Eu, Y:Eu = 80~95 mol%: 5~20 mol%.

4. The application of the upconversion fluorescent nanoprobe according to claim 1 in antibiotic detection, characterized in that, The specific application is: its application in the detection of antibiotic pollution in water bodies.

5. The application of the upconversion fluorescent nanoprobe according to claim 1 in antibiotic detection, characterized in that, The application process includes the following steps: Step 1: Mix the upconversion fluorescent nanoprobe with antibiotic standard solutions of varying concentrations and perform fluorescence detection to establish a linear relationship between the emission intensity of the antibiotic standard solutions of varying concentrations and the upconversion fluorescent nanoprobe. Step 2: Mix the upconversion fluorescent nanoprobe with the antibiotic sample to be tested and then perform fluorescence detection to obtain the emission intensity of the upconversion fluorescent nanoprobe to be tested; Step 3: Substitute the emission intensity of the upconversion fluorescent nanoprobe to be detected into the linear relationship between the concentration gradient of the antibiotic standard solution and the emission intensity of the upconversion fluorescent nanoprobe, and calculate the concentration of antibiotic in the antibiotic sample to be detected.

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