Magnetic fluorescent gold nanocluster molecularly imprinted polymer, preparation method therefor, and use thereof

Abstract

The present invention belongs to the technical field of analytical chemistry. Specifically disclosed are a magnetic fluorescent gold nanocluster molecularly imprinted polymer, a preparation method therefor, and use thereof in sulfadiazine fluorescence detection. The surface of a Fe3O4 nanoparticle is coated with a silica shell layer, and the shell layer is subjected to surface modification and then combined with gold nanoclusters (AuNCs). On this basis, the molecular imprinting technology is employed, wherein sulfadiazine (SD) is selected to serve as a template molecule, and a mixed silane coupling agent (APTES) is used as a functional monomer to coat the surface of Fe3O4@SiO2@AuNCs with a molecular imprinting layer, thus preparing a magnetic fluorescent gold nanocluster imprinted polymer Fe3O4@SiO2@AuNCs-MIPs. The present invention, by integrating the magnetic separation technology, the high selectivity, simplicity, and rapidity of the molecularly imprinted polymer, and the high sensitivity of fluorescence detection, enables accurate and specific recognition of sulfadiazine and can be effectively applied to the analysis and detection of sulfadiazine.

Description

A magnetic fluorescent gold nanocluster molecularly imprinted polymer, its preparation method and application

[0001] This application claims priority to application number 202411414160.0 filed on October 11, 2024, entitled "A magnetic fluorescent gold nanocluster molecularly imprinted polymer and its preparation method and application", the original receiving agency of which is in China. Technical Field

[0002] This invention belongs to the field of analytical chemistry technology, specifically relating to a magnetic fluorescent gold nanocluster molecularly imprinted polymer, its preparation method, and its application. Background Technology

[0003] Sulfadiazine, a type of sulfonamide antibiotic, is widely used due to its excellent antibacterial properties. Currently, sulfadiazine is extensively used for the prevention and treatment of epidemic meningitis, and can also be used to treat respiratory infections and local soft tissue infections caused by bacteria. Due to its low cost and broad antibacterial spectrum, sulfadiazine is widely used in aquaculture. However, because of its stable structure and difficulty in natural degradation, sulfadiazine often leaves excessive residues in water and soil, thus harming the ecological environment and potentially entering the human body through the ecological cycle, posing a threat to human health.

[0004] Currently, the main methods for detecting sulfonamide antibiotics include high-performance liquid chromatography (HPLC), electrochemical detection, and immunoassay. However, these techniques generally suffer from drawbacks such as high cost, complex instruments, and long training times for operators. Therefore, it is crucial to develop a simple, rapid, and efficient detection technique that can be used in complex matrices. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a magnetic fluorescent gold nanocluster molecularly imprinted polymer, and to use the prepared magnetic fluorescent gold nanocluster molecularly imprinted polymer for the efficient and accurate identification and detection of sulfadiazine in aquatic environments.

[0006] To achieve the above objectives, the present invention employs the following technical solution: a method for preparing a magnetic fluorescent gold nanocluster molecularly imprinted polymer, comprising the following steps:

[0007] S1. Fe3O4 nanoparticles were coated with silica using the sol-gel method to obtain Fe3O4 nanoparticles with silica shells, denoted as Fe3O4@SiO2.

[0008] S2. Fe3O4@SiO2 was dispersed in anhydrous ethanol, and then 3-aminopropyltriethoxysilane was added for surface modification to obtain amino-functionalized Fe3O4@SiO2, denoted as Fe3O4@SiO2-NH2.

[0009] S3. Fe3O4@SiO2-NH2 was combined with gold nanoclusters using a surface modification method to obtain Fe3O4@SiO2@AuNCs;

[0010] S4. Using molecular imprinting technology, with sulfadiazine as the template molecule and a mixed silane coupling agent as the functional monomer, a molecular imprinted layer is coated on the surface of Fe3O4@SiO2@AuNCs to prepare magnetic fluorescent gold nanocluster imprinted polymers Fe3O4@SiO2@AuNCs-MIPs.

[0011] As a further improvement to the preparation method of the above-mentioned magnetic fluorescent gold nanocluster molecularly imprinted polymer:

[0012] Preferably, the preparation method of Fe3O4@SiO2 in step S1 is as follows: Fe3O4 nanoparticles are added to an aqueous solution of HCl with a concentration of 0.1-0.3M, with an addition amount of 0.01-0.03 g / ml. After ultrasonic dispersion for 20-50 minutes, the nanoparticles are separated and washed to obtain surface-modified Fe3O4 nanoparticles. Anhydrous ethanol, distilled water, ammonia, and tetraethyl orthosilicate are mixed evenly in a volume ratio of 32:8:1:1 to obtain a mixed solution. 200-220 ml of the mixed solution is added to 400-600 mg of surface-modified Fe3O4 nanoparticles and stirred overnight. The product is separated, washed with distilled water, and vacuum dried to constant weight. The black powder obtained is Fe3O4 nanoparticles coated with a silica shell, denoted as Fe3O4@SiO2.

[0013] Preferably, the specific preparation method of Fe3O4@SiO2-NH2 in step S2 is as follows: Fe3O4@SiO2 is dispersed in anhydrous ethanol at a concentration of 0.01-0.03 g / ml, and then 0.05-0.1 g / ml of 3-aminopropyltriethoxysilane is added to the anhydrous ethanol. The mass ratio of Fe3O4@SiO2 to 3-aminopropyltriethoxysilane is 1:(4.5-5). After stirring and reacting under a protective atmosphere for 10-15 hours, the mixture is separated, washed with ethanol, and dried to obtain amino-functionalized Fe3O4@SiO2, denoted as Fe3O4@SiO2-NH2.

[0014] Preferably, the specific preparation method of Fe3O4@SiO2@AuNCs in step S3 is as follows:

[0015] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was dissolved in 0.1 mM morpholine ethanesulfonic acid (pH 7.0) to a concentration of 0.5-2 mg / mL to obtain a hydrochloride solution. N-hydroxysuccinimide was dissolved in distilled water to a concentration of 0.5-2 mg / mL to obtain an N-hydroxysuccinimide solution. 90 mL of ultrapure water and 4 mL of 40-60 mM chloroauric acid tetrahydrate were mixed and stirred thoroughly to obtain solution 1. 80-100 mg of L-reduced glutathione (GSH) was dissolved in 4 mL of ultrapure water to obtain solution 2. Solution 2 was added to solution 1, heated to 60-80°C and stirred vigorously. After the solution turned slightly yellow, 1 mL of a 28-32% (w / v) threonine (Thr) aqueous solution was added, and the reaction proceeded. The product should be left overnight, and then dialyzed using a dialysis bag for 20-25 hours to obtain a solution containing carboxyl-functionalized gold nanoclusters (AuNCs). The hydrochloride solution is mixed with the solution containing carboxyl-functionalized gold nanoclusters (AuNCs), and then N-hydroxysuccinimide solution is added in a volume ratio of 6:10:6. The mixture is stirred to obtain a final mixture. 50-70 mg of Fe3O4@SiO2-NH2 and 0.05-0.2 mM morpholine ethanesulfonic acid (pH 5-8) are added to the mixture, with the volume ratio of morpholine ethanesulfonic acid to the solution containing carboxyl-functionalized gold nanoclusters (AuNCs) being 5:1. After ultrasonic dispersion, the mixture is stirred overnight in the dark and separated. The final product is washed with a 0.01 M phosphate buffer solution (pH 7.0) to obtain Fe3O4@SiO2@AuNCs.

[0016] Preferably, the preparation method of Fe3O4@SiO2@AuNCs-MIPs in step S4 is as follows: 80-150 mg of Fe3O4@SiO2@AuNCs nanoparticles are added to 30-50 ml of phosphate buffer solution and mixed evenly. The concentration of the phosphate buffer solution is 10 mmol / L. -1 The pH was set to 7.0. Then, 0.15-0.2 mmol of a mixed silane coupling agent was added, along with 10 mg of sulfadiazine as the template molecule. After stirring evenly, 60-100 μl of tetraethyl orthosilicate and 60-100 μl of ammonia water were added. The molar ratio of the template molecule to the mixed silane coupling agent was 1:4. The mixture was stirred continuously at room temperature for 12-20 h. After the reaction was completed, the solid product was separated by centrifugation. Then, it was repeatedly washed with a mixed solvent of methanol and acetic acid in a volume ratio of 9:1 until the supernatant could not be detected by a UV spectrophotometer. Finally, it was vacuum dried to obtain the magnetic fluorescent gold nanocluster imprinted polymer, denoted as Fe3O4@SiO2@AuNCs-MIPs.

[0017] Preferably, in step S1, the black powder Fe3O4@SiO2 is dried in a vacuum drying oven at a temperature of 40-60°C, and the separated product is washed sequentially with distilled water and anhydrous ethanol; in steps S1 and S2, the reaction products are separated by adsorption separation using a strong magnet.

[0018] Preferably, the mixed silane coupling agent mentioned in step S4 is APTES.

[0019] A second objective of this invention is to provide a magnetic fluorescent gold nanocluster molecularly imprinted polymer prepared by any of the above-described preparation methods.

[0020] A third objective of this invention is to provide an application of the above-mentioned magnetic fluorescent gold nanocluster molecularly imprinted polymer in the detection of sulfadiazine.

[0021] As a further improvement to the application of the aforementioned magnetic fluorescent gold nanocluster molecularly imprinted polymer in the detection of sulfadiazine:

[0022] Preferably, the aqueous solution of the magnetic fluorescent gold nanocluster molecularly imprinted polymer is mixed with sulfadiazine, and the changes in fluorescence intensity before and after mixing are compared. The advantages of this invention compared to the prior art are as follows:

[0023] 1) Electrons within metal nanoclusters can transfer between energy spectrum segments, generating absorption and emission spectra, and exhibiting photoluminescence with excellent fluorescence stability and high luminescence efficiency. This superior luminescence performance makes them widely used in ion detection, fluorescence detection, and bioanalysis. Molecular imprinting technology is a method of synthesizing polymers with specific recognition capabilities for target analytes through chemical methods. Molecularly imprinted polymers possess numerous advantages, including specific recognition, environmental stability, convenient synthesis, and relatively low preparation costs. Combining gold nanoclusters with molecularly imprinted polymers can form a fluorescent molecularly imprinted complex with excellent optical properties and selective recognition capabilities.

[0024] This invention utilizes a magnetic nanomaterial as the core, coated with a silicon layer, providing a larger surface area and better dispersibility compared to other bare magnetic materials. This shell-core structured magnetic molecularly imprinted material completely embeds magnetic particles, preventing leakage. The recognition sites are located on the surface of polymer microspheres, resulting in higher binding and separation efficiencies for the target analyte. Simultaneously, this material exhibits superparamagnetism, enabling rapid magnetic separation under an external magnetic field, allowing for material recycling.

[0025] 2) In step S2, 3-aminopropyltriethoxysilane is a mixed silane coupling agent, sulfadiazine (SD) is a template molecule, tetraethyl orthosilicate is a crosslinking agent, and ammonia is an initiator.

[0026] This invention combines the advantages of high selectivity and specificity of molecular imprinting technology, high specific surface area of ​​mesoporous materials, and superior optical properties of gold nanoclusters (AuNCs), and integrates the advantages of gold nanoclusters, molecularly imprinted polymers, and magnetic materials to synthesize a fluorescent molecularly imprinted polymer material with an ordered mesoporous structure that has high efficiency and specificity.

[0027] This invention characterizes the synthesized molecularly imprinted polymer using FT-IR and structural characterization. It investigates the effects of quenching time, template molecule concentration, template solution at different pH values, and actual samples on the detection of sulfadiazine by the fluorescently imprinted polymer. The optimal detection conditions were obtained, and all yielded good results. This method features a simple synthesis process and efficient template molecule elution. The synthesized material exhibits stable chemical and physical properties and can detect sulfadiazine with high specificity. Attached Figure Description

[0028] Figure 1 is a schematic diagram of the preparation process of molecularly imprinted microspheres (MIPs) with mesoporous structures;

[0029] Figure 2 shows the infrared spectra of Fe3O4@SiO2 prepared in Example 1, Fe3O4@SiO2@AuNCs prepared in Example 2, and Fe3O4@SiO2@AuNCs-MIPs prepared in Example 3.

[0030] Figure 3 is a SEM image of Fe3O4@SiO2 prepared in Example 1 and Fe3O4@SiO2@AuNCs-MIPs prepared in Example 3;

[0031] Figure 4 is a magnetization curve of Fe3O4@SiO2@AuNCs-MIPs prepared in Example 3;

[0032] Figure 5 shows the change in fluorescence intensity of Fe3O4@SiO2@AuNCs-MIPs prepared in Example 3 as a function of time;

[0033] Figure 6 shows the fluorescence quenching response time of Fe3O4@SiO2@AuNCs-MIPs prepared in Example 3 to sulfadiazine.

[0034] Figure 7 shows the effect of sulfadiazine on the fluorescence quenching of Fe3O4@SiO2@AuNCs-MIPs at different pH values;

[0035] Figure 8 shows the effect of different concentrations of template molecules on the fluorescence intensity of Fe3O4@SiO2@AuNCs-MIPs;

[0036] Figure 9 shows the changes in fluorescence quenching of Fe3O4@SiO2@AuNCs-MIPs and Fe3O4@SiO2@AuNCs-MIPs by four sulfonamide antibiotics. Embodiments of the present invention

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. Example 1

[0038] This embodiment provides a method for preparing Fe3O4@SiO2-NH2, which specifically includes the following steps:

[0039] S1. Add 2.0g of Fe3O4 to 100mL of 0.1M HCl aqueous solution and ultrasonically disperse for 30 minutes to modify the surface of Fe3O4. Wash the separated Fe3O4 powder several times with distilled water and ethanol to obtain surface-modified Fe3O4 nanoparticles.

[0040] S2. Anhydrous ethanol (160 mL), distilled water (40 mL), ammonia (5 mL), and tetraethyl orthosilicate (5 mL) were vigorously stirred until homogeneous to obtain a mixed solution. The mixed solution was added to the surface-modified Fe3O4 (500 mg) nanoparticles and stirred overnight. The product was separated by adsorption with a strong magnet, washed repeatedly with distilled water, and vacuum dried at 50 °C to constant weight. The black powder obtained was Fe3O4 nanoparticles coated with a silica shell, denoted as Fe3O4@SiO2.

[0041] S3. Take 1 g of Fe3O4@SiO2 and disperse it in anhydrous ethanol (50 mL) to a concentration of 0.02 g / mL. Then add 3-aminopropyltriethoxysilane (5 mL, 4.82 g). The amount of 3-aminopropyltriethoxysilane added to anhydrous ethanol is 0.096 g / mL. The mass ratio of Fe3O4@SiO2 to 3-aminopropyltriethoxysilane is 1:4.82. Stir under a nitrogen atmosphere for 12 hours until the reaction is complete. Separate the synthesized product by adsorption with a strong magnet. Wash repeatedly with ethanol and dry in a vacuum oven to constant weight to obtain Fe3O4@SiO2-NH2. Example 2

[0042] This embodiment provides a Fe3O4@SiO2@AuNC s The preparation method of [the substance] specifically includes the following steps:

[0043] S1. Dissolve 20 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 20 mL of 0.1 mM morpholine ethanesulfonic acid (pH=7.0) to obtain a hydrochloride solution with a concentration of 1 mg / mL.

[0044] 10 mg of N-hydroxysuccinimide was dissolved in 10 mL of distilled water to obtain an N-hydroxysuccinimide solution with a concentration of 1 mg / mL.

[0045] 90 mL of ultrapure water and 4 mL of 50 mM chloroauric acid tetrahydrate were mixed and stirred until homogeneous to form solution 1; 92 mg of L-reduced glutathione (GSH) was dissolved in 4 mL of ultrapure water to form solution 2; solution 2 was added to solution 1, heated to 70 °C and stirred vigorously. After the solution turned slightly yellow, 1 mL of threonine (Thr) aqueous solution (concentration 30%, w / v) was added, and the reaction was allowed to proceed overnight. The product was dialyzed through a dialysis bag for 24 h to obtain a solution containing carboxyl-functionalized gold nanoclusters (AuNCs).

[0046] S2. Take 6 mL of hydrochloric acid solution and mix it with 10 mL of solution containing carboxyl-functionalized gold nanoclusters AuNCs. Stir for 10 minutes, then add 6 mL of N-hydroxysuccinimide solution and stir for 10 minutes to obtain a mixture.

[0047] S3. Add 60 mg of Fe3O4@SiO2-NH2 prepared in Example 1 and 50 ml of 0.1 mM morpholine ethanesulfonic acid (pH=6) to the mixture. After ultrasonic dispersion, stir overnight in the dark and separate. Wash three times with 0.01 M phosphate buffer solution (pH 7.0) to remove unreacted Thr-AuNCs and obtain Fe3O4@SiO2@AuNCs. Example 3

[0048] This embodiment provides a method for preparing Fe3O4@SiO2@AuNCs-MIPs, which specifically includes the following steps:

[0049] S1. Take 100 mg of Fe3O4@SiO2@AuNCs nanoparticles prepared in Example 2 and add them to 40 ml of phosphate buffer solution (10 mmol / L). -1 Mix thoroughly in a solution containing water at pH 7.0.

[0050] S2. Add 0.16 mmol of mixed silane coupling agent (APTES) and 10 mg (0.04 mmol) of sulfadiazine as template molecules. After stirring for 30 min, add 80 μL of tetraethyl orthosilicate and 100 μL of ammonia. Continue stirring at room temperature for 16 h. After the reaction is complete, centrifuge the solution to separate the solid product.

[0051] S3. The separated solid product is repeatedly washed with a mixed solvent of methanol and acetic acid (v / v=9:1) until the template molecule sulfadiazine is no longer detectable by a UV spectrophotometer. Then, it is vacuum dried to obtain the magnetic fluorescent gold nanocluster imprinted polymer, denoted as Fe3O4@SiO2@AuNCs-MIPs. Comparative Example

[0052] This comparative example provides a method for preparing Fe3O4@SiO2@AuNCs-NIPs, which specifically includes the following steps:

[0053] S1. Take 100 mg of Fe3O4@SiO2@AuNCs nanoparticles prepared in Example 2 and add them to 40 ml of phosphate buffer solution (10 mmol / L). -1 Mix thoroughly in a solution containing water at pH 7.0.

[0054] S2. Add 0.16 mmol of mixed silane coupling agent (APTES), stir for 30 min, then add 80 μL of tetraethyl orthosilicate and 100 μL of ammonia water, and continue stirring at room temperature for 16 h. After the reaction is complete, centrifuge the solution to separate the solid product.

[0055] S3. The separated solid product was repeatedly washed with a mixed solvent of methanol and acetic acid (v / v=9:1) until the supernatant could not be detected by a UV spectrophotometer. Then it was vacuum dried to obtain a magnetic fluorescent gold nanocluster imprinted polymer, denoted as Fe3O4@SiO2@AuNCs-NIPs, without the addition of the template molecule sulfadiazine.

[0056] 1. Preparation process

[0057] Figure 1 is a flowchart of the preparation of Fe3O4@SiO2@AuNCs-MIPs polymer according to the present invention. As shown in Figure 1, the present invention first coats Fe3O4 nanoparticles with a silica shell and modifies the surface of the shell to obtain Fe3O4 nanoparticles with a silica shell, namely Fe3O4@SiO2; then Fe3O4@SiO2 is combined with gold nanoclusters (AuNCs), and molecular imprinting technology is used to select sulfadiazine (SD) as a template molecule and mixed silane coupling agent (APTES) as a functional monomer to coat the surface of Fe3O4@SiO2@AuNCs with a molecular imprinting layer, thus preparing the magnetic fluorescent gold nanocluster imprinted polymer Fe3O4@SiO2@AuNCs-MIPs.

[0058] 2. Fourier Transform Infrared (FT-IR) Characterization of Molecularly Imprinted Microspheres

[0059] Figure 2 shows the Fourier transform infrared (FT-IR) characterization diagrams of three materials: Fe3O4@SiO2 prepared in Example 1, Fe3O4@SiO2@AuNCs prepared in Example 2, and Fe3O4@SiO2@AuNCs-MIPs prepared in Example 3, as shown in Figure 2(a), (b), and (c) respectively.

[0060] Figure (a) at 579cm -1 There is a distinct absorption peak at 1562 cm⁻¹, which is due to the vibration of the Fe-O bond; additionally, there is an absorption peak at 1562 cm⁻¹. -1 and 3421cm -1 The two wave peaks correspond to the bending and stretching vibrations of hydrogen bonds, respectively. At 1039 cm⁻¹ -1 The characteristic peak is attributed to the vibration of Si-O-Si, 771 cm⁻¹. -1 The characteristic peak at 2947 cm⁻¹ is attributed to the vibration of Si-O; furthermore, the peak at 2947 cm⁻¹ is also attributed to the vibration of Si-O. −1 The peak at 1400 cm⁻¹ may be attributed to the hydrocarbon stretching vibration of 3-aminopropyltriethoxysilane, thus the Fe₃O₄@SiO₂ surface is modified with amino functionalization. From Figure (b), the peak at 1400 cm⁻¹ of the CO-NH vibration... -1 and 1495cm -1 The peak value at 574 cm⁻¹ indicates that the gold nanoclusters successfully bonded to the silica on the Fe₃O₄ surface via CO-NH bonds. (See Figure (c) for the peak value at 574 cm⁻¹.) −1 The peak value at that point may be related to silicon dioxide; therefore, the infrared spectrum confirms the successful synthesis of molecularly imprinted microspheres.

[0061] 3. Scanning electron microscopy (SEM) characterization

[0062] A small amount of Fe3O4@SiO2 prepared in Example 1 and the magnetic fluorescent gold nanocluster imprinted polymer Fe3O4@SiO2@AuNCs-MIPs prepared in Example 3 were uniformly spread on gold sheets, and then the morphology of the samples was observed by scanning electron microscopy. The scanning electron microscopy images are shown in Figure 3(a) and (b) respectively.

[0063] Figure (a) shows that the Fe3O4@SiO2 particles are uniform in size and regularly spherical, with particle size concentrated between 130-210 nm; Figure (b) shows that the Fe3O4@SiO2@AuNCs-MIPs particles are irregularly spherical and vary in size, ranging from 260-550 nm, with a particle size significantly larger than Fe3O4@SiO2, resulting in the surface polymer imprint layer being anchored to the surface of Fe3O4@SiO2.

[0064] 4. Magnetization curve (VSM)

[0065] The magnetic properties of the Fe3O4@SiO2@AuNCs-MIPs prepared in Example 3 were determined using a comprehensive physical property measurement system.

[0066] Figure 4 shows the magnetization curves of Fe3O4@SiO2@AuNCs-MIPs at room temperature. It can be seen that this magnetic material exhibits superparamagnetism; even with a molecularly imprinted layer and a silica core coating the Fe3O4 core, the composite does not show magnetic hysteresis. Therefore, this composite possesses excellent magnetic properties and can be used for magnetic adsorption separation.

[0067] 5. Study on the fluorescence properties of molecularly imprinted microspheres

[0068] Weigh the Fe3O4@SiO2@AuNCs-MIPs polymer powder obtained in Example 3, add it to deionized water to prepare a 500 mg / L fluorescently imprinted polymer standard solution, and store it at 4°C in the dark for later use.

[0069] 1) Stability test

[0070] Take an appropriate amount of the above-mentioned fluorescently imprinted polymer standard solution, shake it evenly at 25°C, measure the fluorescence value of the solution once every 0 minutes, and then test and record the fluorescence intensity every two minutes with a fluorescence spectrophotometer (within 1 hour). Each sample is measured in parallel 3 times, and the average value is taken.

[0071] Figure 5 shows the change in fluorescence intensity of Fe3O4@SiO2@AuNCs-MIPs over time. At room temperature, after shaking for 2 minutes every 60 minutes, fluorescence was rapidly detected. The fluorescence of Fe3O4@SiO2@AuNCs-MIPs remained relatively stable. This indicates that the prepared MIPs possess excellent fluorescence stability.

[0072] 2) Study on quenching response time

[0073] Take an appropriate amount of the above fluorescently imprinted polymer standard solution, add 10 ml of 20 mg / L sulfadiazine standard solution and shake well. Measure the fluorescence value of the solution once every 0 minutes, and then test and record the fluorescence intensity every two minutes with a fluorescence spectrophotometer (30 min, each sample was measured in parallel 3 times, and the average value was taken).

[0074] Figure 6 shows the change in fluorescence quenching response of Fe3O4@SiO2@AuNCs-MIPs to sulfadiazine over time. When 10 ml of 20 mg / L sulfadiazine was added, Fe3O4@SiO2@AuNCs-MIPs specifically bound to the template molecule through the specific imprinted cavity and quenched within a short time. The fluorescence intensity of the solution gradually decreased and reached equilibrium after about 16 min. Therefore, the optimal response time of this fluorescently imprinted polymer to sulfadiazine is 16 min.

[0075] 3) Effect of different pH values ​​on fluorescence quenching of Fe3O4@SiO2@AuNCs-MIPs

[0076] The pH of the solution system was adjusted using phosphate buffer solution (PBS buffer solution), and fluorescence quenching experiments were conducted within the pH range of 3-12.

[0077] Prepare a 100 mg / L sulfadiazine standard solution using acetonitrile / water (v / v=1:1) as the solvent and store it at 4°C in the dark.

[0078] Take 1.5 ml of the above fluorescently imprinted polymer standard solution and place it into multiple centrifuge tubes. Add 5 ml of sulfadiazine standard solution to each centrifuge tube, and then add PBS buffer solutions of different pH values ​​to each tube to make up to 10 ml, resulting in multiple solutions with pH values ​​of 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Shake and mix well at room temperature, let stand for 16 min, and then measure the fluorescence using a fluorescence spectrophotometer. Each sample is measured in triplicate, and the average value is taken.

[0079] Figure 7 shows the effect of sulfadiazine on the fluorescence quenching of Fe3O4@SiO2@AuNCs-MIPs at different pH values. When pH < 6, the fluorescence quenching effect of Fe3O4@SiO2@AuNCs-MIPs increases rapidly with increasing pH. Within the pH range of 6.0-8.0, the increase in fluorescence quenching effect with increasing pH is relatively slow, reaching a peak at pH = 8.0. Its fluorescence quenching performance remains relatively stable between pH 6.0-9.0. When pH > 9, the fluorescence quenching effect gradually weakens, but remains higher than in acidic environments. However, antibiotics are destroyed under strong acid and strong alkali conditions, and these conditions also affect the fluorescence signal. Therefore, pH 8.0 is the optimal pH.

[0080] 4) Effect of different concentrations of template molecules on the fluorescence intensity of Fe3O4@SiO2@AuNCs-MIPs

[0081] Prepare a 200 mg / L sulfadiazine standard solution using acetonitrile / water (v / v=1:1) as the solvent, and dilute it to prepare sulfadiazine standard solutions of different concentrations (0-25 mg / L). Store at 4°C in the dark.

[0082] 1.5 mL of fluorescently imprinted polymer standard solution was added to 6 centrifuge tubes and diluted with an equal volume of PBS buffer solution at pH 8.0. The solutions were mixed evenly to prepare sulfadiazine standard solutions of different concentrations of 0, 5, 10, 15, 20, and 25 mg / L. 5 mL of each of the different concentrations of sulfadiazine standard solutions was taken and shaken at room temperature for 16 minutes. The fluorescence emission spectrum of each sulfadiazine standard solution was then measured and recorded.

[0083] The Fe3O4@SiO2@AuNCs-NIPs prepared in the comparative example were subjected to the same experimental procedures as Fe3O4@SiO2@AuNCs-MIPs to compare the effects of different concentrations of template molecules on the fluorescence intensity of Fe3O4@SiO2@AuNCs-NIPs. Figure 8 shows the effect of different concentrations of template molecules on the fluorescence intensity of Fe3O4@SiO2@AuNCs-MIPs. In Figure 8(a), after adding different concentrations of sulfadiazine SD from 0 to 25 mg / L, the fluorescence intensity decreased continuously with the increase of concentration. Compared with Figure 8(b), it was found that the decrease in fluorescence intensity of Fe3O4@SiO2@AuNCs-MIPs was greater than that of Fe3O4@SiO2@AuNCs-NIPs. This is because there are specific recognition pores in Fe3O4@SiO2@AuNCs-MIPs that correspond to the template molecule sulfadiazine in space and size, which can be adsorbed through hydrogen bonds. Therefore, the adsorption of sulfadiazine SD by Fe3O4@SiO2@AuNCs-MIPs is greater than the non-specific adsorption of sulfadiazine SD by Fe3O4@SiO2@AuNCs-NIPs.

[0084] 5) Competitive selection experiment of Fe3O4@SiO2@AuNCs-MIPs

[0085] In the selective competition experiment, three sulfonamide antibiotics were selected as structural analogs for comparison. The three structural analogs were sulfathiazole, sulfadimethoxypyrimidine, and sulfapyridine. 10 mg of each of the above sulfathiazole, sulfadimethoxypyrimidine, and sulfapyridine were weighed and prepared into a 200 mg / L standard stock solution using acetonitrile / water (v:v=1:1) as the solvent. A measured amount of the stock solution was then diluted to prepare a 20 mg / L standard solution, which was then stored in a sealed refrigerator for later use.

[0086] Then, 1.5 mL of Fe3O4@SiO2@AuNCs-MIPs (500 mg / L) standard solution was added to a centrifuge tube, diluted with an equal volume of pH 8.0 PBS buffer, and thoroughly mixed. A series of prepared standard solutions of several sulfonamide antibiotics of the same concentration were added in equal volumes to the centrifuge tubes, and the mixtures were shaken at room temperature for 16 minutes. The fluorescence emission spectra of the mixed solutions were recorded. The Fe3O4@SiO2@AuNCs-NIPs prepared in the comparative experiment were processed using the same procedure as a control.

[0087] Figure 9 shows the fluorescence quenching of Fe3O4@SiO2@AuNCs-MIPs and Fe3O4@SiO2@AuNCs-MIPs by four sulfonamide antibiotics. The figure shows that sulfadiazine (SD) exhibits the greatest quenching effect on Fe3O4@SiO2@AuNCs-MIPs. While sulfathiazole and sulfapyridine are structurally similar to sulfadiazine, they differ in their matching cavity structures, resulting in lower fluorescence quenching. Sulfamethoxypyrimidine, due to its greatest structural difference from sulfadiazine, exhibits the least fluorescence quenching. Since Fe3O4@SiO2@AuNCs-NIPs do not form corresponding cavity structures and cannot specifically bind, the other three sulfonamide antibiotics show little difference in their fluorescence quenching effects on Fe3O4@SiO2@AuNCs-NIPs.

[0088] 6) Analysis of real water samples

[0089] Water from the artificial lake at Anhui University was used as the actual sample and pretreated. The water sample was left to stand overnight, and the supernatant was collected and filtered through a disposable filter (0.22 μm) to obtain the sample solution. Sulfadiazine standard solution with a concentration of 20 mg / L was added to the sample solution until the concentration of sulfadiazine was 1 mg / L, 5 mg / L and 10 mg / L respectively, and the solution was stored in a refrigerator.

[0090] Table 1 Recovery rates of Fe3O4@SiO2@AuNCs-MIPs in actual samples

[0091] Water sampleAdded(mg / L)Found(mg / L)RecoveryRSD(N=3,%)111.0401104.04.2255.117102.33.73109.89198.95.9

[0092] As shown in Table 1, a good recovery rate was obtained within the linear fitting range. The recovery rate was between 98.9% and 104.0%, with a relative standard deviation of <5.9%. This magnetic molecularly imprinted fluorescence sensing system exhibited excellent performance in measuring real samples, and its fluorescence detection of the template molecule sulfadiazine (SD) was accurate and effective. Therefore, it can be used to detect sulfadiazine (SD) in real-world environments.

[0093] Those skilled in the art should understand that the above descriptions are merely several specific embodiments of the present invention, and not all embodiments. It should be noted that many modifications and improvements can be made by those skilled in the art, and all modifications or improvements not exceeding the scope of the claims should be considered within the protection scope of the present invention.

Claims

1. A method for preparing a magnetic fluorescent gold nanocluster molecularly imprinted polymer, characterized in that, Includes the following steps: S1. Fe3O4 nanoparticles were coated with silica using the sol-gel method to obtain Fe3O4 nanoparticles with silica shell, denoted as Fe3O4@SiO2. S2. Fe3O4@SiO2 was dispersed in anhydrous ethanol, and then 3-aminopropyltriethoxysilane was added for surface modification to obtain amino-functionalized Fe3O4@SiO2, denoted as Fe3O4@SiO2-NH2. S3. Fe3O4@SiO2-NH2 was combined with gold nanoclusters using a surface modification method to obtain Fe3O4@SiO2@AuNCs; S4. Using molecular imprinting technology, with sulfadiazine as the template molecule and a mixed silane coupling agent as the functional monomer, a molecular imprinted layer is coated on the surface of Fe3O4@SiO2@AuNCs to prepare magnetic fluorescent gold nanocluster imprinted polymers Fe3O4@SiO2@AuNCs-MIPs.

2. The method for preparing the magnetic fluorescent gold nanocluster molecularly imprinted polymer according to claim 1, characterized in that, The preparation method of Fe3O4@SiO2 in step S1 is as follows: Fe3O4 nanoparticles are added to an aqueous solution of 0.1-0.3M HCl at a concentration of 0.01-0.03 g / ml. After ultrasonic dispersion for 20-50 minutes, the nanoparticles are separated and washed to obtain surface-modified Fe3O4 nanoparticles. Anhydrous ethanol, distilled water, ammonia, and tetraethyl orthosilicate are mixed evenly in a volume ratio of 32:8:1:1 to obtain a mixed solution. 200-220 ml of the mixed solution is added to 400-600 mg of surface-modified Fe3O4 nanoparticles and stirred overnight. The product is separated, washed with distilled water, and vacuum dried to constant weight. The black powder obtained is Fe3O4 nanoparticles coated with a silica shell, denoted as Fe3O4@SiO2.

3. The method for preparing the magnetic fluorescent gold nanocluster molecularly imprinted polymer according to claim 1, characterized in that, The specific preparation method of Fe3O4@SiO2-NH2 in step S2 is as follows: Fe3O4@SiO2 is dispersed in anhydrous ethanol at a concentration of 0.01-0.03 g / ml. Then, 0.05-0.1 g / ml of 3-aminopropyltriethoxysilane is added to the anhydrous ethanol. The mass ratio of Fe3O4@SiO2 to 3-aminopropyltriethoxysilane is 1:(4.5-5). After stirring and reacting under a protective atmosphere for 10-15 hours, the mixture is separated, washed with ethanol, and dried to obtain amino-functionalized Fe3O4@SiO2, denoted as Fe3O4@SiO2-NH2.

4. The method for preparing the magnetic fluorescent gold nanocluster molecularly imprinted polymer according to claim 1, characterized in that, The specific preparation method of Fe3O4@SiO2@AuNCs in step S3 is as follows: Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 0.1 mM morpholine ethanesulfonic acid (pH 7.0) to obtain a hydrochloride solution (0.5-2 mg / mL). Dissolve N-hydroxysuccinimide in distilled water to obtain an N-hydroxysuccinimide solution (0.5-2 mg / mL). Mix 90 mL of ultrapure water with 4 mL of 40-60 mM chloroauric acid tetrahydrate and stir until homogeneous to obtain solution 1. Dissolve 80-100 mg of L-reduced glutathione (GSH) in 4 mL of ultrapure water to obtain solution 2. Add solution 2 to solution 1, heat to 60-80°C and stir vigorously until the solution turns slightly yellow. Then add 1 mL of a solution with a concentration of 28-32 mg / mL. A % (w / v) aqueous solution of threonine (Thr) was reacted overnight. The product was dialyzed using a dialysis bag for 20-25 hours to obtain a solution containing carboxyl-functionalized gold nanoclusters (AuNCs). The hydrochloride solution was mixed with the solution containing carboxyl-functionalized gold nanoclusters (AuNCs), and then N-hydroxysuccinimide solution was added. The volume ratio of the three was 6:10:6, and the mixture was stirred to obtain a mixture. 50-70 mg of Fe3O4@SiO2-NH2 and 0.05-0.2 mM morpholine ethanesulfonic acid (pH 5-8) were added to the mixture. The volume ratio of the added morpholine ethanesulfonic acid to the volume of the solution containing carboxyl-functionalized gold nanoclusters (AuNCs) was 5:

1. After ultrasonic dispersion, the mixture was stirred overnight in the dark and separated. The mixture was washed with a 0.01 M phosphate buffer solution (pH 7.0) to obtain Fe3O4@SiO2@AuNCs.

5. The method for preparing the magnetic fluorescent gold nanocluster molecularly imprinted polymer according to claim 1, characterized in that, The preparation method of Fe3O4@SiO2@AuNCs-MIPs in step S4 is as follows: 80-150 mg of Fe3O4@SiO2@AuNCs nanoparticles are added to 30-50 ml of phosphate buffer solution and mixed thoroughly. The concentration of the phosphate buffer solution is 10 mmol / L. -1 The pH was set to 7.

0. Then, 0.15-0.2 mmol of a mixed silane coupling agent was added, along with 10 mg of sulfadiazine as the template molecule. After stirring evenly, 60-100 μl of tetraethyl orthosilicate and 60-100 μl of ammonia water were added. The molar ratio of the template molecule to the mixed silane coupling agent was 1:

4. The mixture was stirred continuously at room temperature for 12-20 h. After the reaction was completed, the solid product was separated by centrifugation. Then, it was repeatedly washed with a mixed solvent of methanol and acetic acid in a volume ratio of 9:1 until the supernatant could not be detected by a UV spectrophotometer. Finally, it was vacuum dried to obtain the magnetic fluorescent gold nanocluster imprinted polymer, denoted as Fe3O4@SiO2@AuNCs-MIPs.

6. The method for preparing the magnetic fluorescent gold nanocluster molecularly imprinted polymer according to claim 2, characterized in that, In step S1, the black powder Fe3O4@SiO2 is dried in a vacuum drying oven at a temperature of 40-60℃. The separated product is washed sequentially with distilled water and anhydrous ethanol. In steps S1 and S2, the reaction products are separated by adsorption using a strong magnet.

7. The method for preparing the magnetic fluorescent gold nanocluster molecularly imprinted polymer according to claim 1, characterized in that, The mixed silane coupling agent mentioned in step S4 is APTES.

8. A magnetic fluorescent gold nanocluster molecularly imprinted polymer prepared by the preparation method according to any one of claims 1-7.

9. The application of the magnetic fluorescent gold nanocluster molecularly imprinted polymer of claim 8 in the fluorescence detection of sulfonamide antibiotics.

10. The application of the magnetic fluorescent gold nanocluster molecularly imprinted polymer according to claim 9 in the fluorescence detection of sulfonamide antibiotics, characterized in that, An aqueous solution of the magnetic fluorescent gold nanocluster molecularly imprinted polymer was mixed with sulfadiazine, and the changes in fluorescence intensity before and after mixing were compared.

Images