Nitrogen and phosphorus co-doped carbon dots@zif-67@mips composite material, preparation method and application thereof

Abstract

The application discloses a nitrogen and phosphorus co-doped carbon dot@ZIF-67@MIPs composite material and a preparation method and application thereof, and belongs to the technical field of detection materials. The composite material is prepared by loading nitrogen and phosphorus co-doped carbon dots on the surface of a cobalt-based metal organic framework ZIF-67 and then performing surface molecular imprinting technology, and has the fluorescence characteristics of the carbon dots, the selective recognition ability of the molecular imprinting and the stability of the ZIF-67. The nitrogen and phosphorus co-doped carbon dots have good fluorescence performance, can undergo electron transfer with sulfonamidopyrimidine and form a hydrogen bond-like action, and the ZIF-67 improves the dispersity and stability of the carbon dots. The material after surface imprinting has imprinting cavities similar in size and shape to sulfonamidopyrimidine, and can selectively recognize sulfonamidopyrimidine. The composite material can be used for fluorescence detection of sulfonamidopyrimidine in actual samples such as milk and environmental water samples.

Description

Technical Field

[0001] This invention belongs to the field of detection material preparation technology, specifically relating to a nitrogen and phosphorus co-doped carbon dot @ZIF-67@MIPs composite material and its preparation method, as well as its application in sulfadiazine detection. Background Technology

[0002] Sulfadiazine (SDZ) is a sulfonamide antibiotic widely used in animal husbandry and aquaculture. It has advantages such as broad-spectrum antibacterial activity and low cost. However, it is slowly metabolized in animals and easily accumulates in humans through the food chain. Long-term intake can lead to health risks such as allergic reactions, increased drug resistance, and intestinal flora imbalance. Therefore, it is essential to establish an analytical method that can accurately determine sulfadiazine in actual samples.

[0003] Currently, the main methods for detecting sulfadiazine include high-performance liquid chromatography (HPLC), high-performance liquid chromatography-mass spectrometry (HPLC-MS), and enzyme-linked immunosorbent assay (ELISA). Among these, HPLC-MS offers high precision and accuracy, but it suffers from drawbacks such as complex sample pretreatment, long detection cycles, expensive equipment, and demanding operational requirements, making it difficult to achieve rapid on-site detection. ELISA is simple to operate and has a relatively fast detection speed, but it suffers from issues such as easy antibody inactivation and high detection costs, making it unsuitable for rapid screening of large batches of samples.

[0004] Fluorescence detection methods have attracted much attention due to their ease of operation and rapid response. Carbon dots, as a novel fluorescent nanomaterial, possess excellent biocompatibility, high stability, and low toxicity, and are widely used in the field of fluorescence detection. Metal-organic frameworks can be used to disperse carbon dots, preventing their aggregation and enhancing material stability. Molecularly imprinted polymers (MIPs) have the ability to recognize target molecules and can effectively eliminate the influence of interfering substances. However, each single material has its limitations in detection performance. Therefore, developing a fluorescence detection method and material that is fast-responding, highly resistant to interference, and highly accurate has significant application value. Summary of the Invention

[0005] One objective of this invention is to address the problems of poor selectivity and susceptibility to matrix interference in the detection of sulfadiazine in real samples (milk, environmental water samples) using existing fluorescent detection materials. This invention provides a nitrogen- and phosphorus co-doped carbon dots@ZIF-67@MIPs composite material (N,P-CDs@ZIF-67@MIPs). This fluorescent nanocomposite material uses nitrogen- and phosphorus co-doped carbon dots as the fluorescence signal source and ZIF-67 as the carrier. A cavity structure with a shape and size similar to sulfadiazine is formed through surface molecular imprinting technology. ZIF-67 improves the dispersibility and structural stability of the carbon dots, while the outer molecular imprinting layer provides recognition sites, effectively reducing interference from structural analogs and matrix components. The synergistic effect of these three components enables quantitative fluorescence detection of sulfadiazine in real samples.

[0006] To achieve the above objectives, the present invention employs the following technical solution: a nitrogen- and phosphorus co-doped carbon dot @ZIF-67@MIPs composite material, wherein the composite material is nitrogen- and phosphorus co-doped carbon dot @ zeolite imidazole ester framework-67 @ molecularly imprinted polymer, and the overall structure is an irregular spherical structure with a particle size of 200-1000 nm; the polymer uses a cobalt-based metal-organic framework ZIF-67 as a carrier, the surface of the carrier is loaded with nitrogen- and phosphorus co-doped carbon dots, and the surface of the carrier and the nitrogen- and phosphorus co-doped carbon dots is coated with a molecularly imprinted polymer layer, wherein the molecularly imprinted polymer layer has specific recognition sites for sulfadiazine.

[0007] Further improvements to the nitrogen and phosphorus co-doped carbon dot @ZIF-67@MIPs composite material: Preferably, the ZIF-67 has a rhombic dodecahedral crystal structure with a particle size of 100-900 nm.

[0008] Preferably, the particle size of the nitrogen and phosphorus co-doped carbon dots is 2-4 nm, and the mass ratio of the carbon dots to the carrier ZIF-67 is (0.0022-0.022):1.

[0009] Preferably, the thickness of the molecularly imprinted polymer layer is 10-40 nm.

[0010] The second objective of this invention is to provide a method for preparing the above-mentioned nitrogen and phosphorus co-doped carbon dot @ZIF-67@MIPs composite material, comprising the following steps: S1. Prepare a nitrogen-phosphorus co-doped carbon dot solution, i.e., N,P-CDs solution, by hydrothermal method using citric acid, ethylenediamine and phosphoric acid. S2. ZIF-67 powder was prepared by cobalt nitrate hexahydrate and 2-methylimidazole through a coordination reaction, and then dispersed in a solvent to obtain ZIF-67 dispersion. S3. Mix the N,P-CDs solution with the ZIF-67 dispersion evenly, add the template molecules sulfadiazine, 3-aminopropyltriethoxysilane, tetraethyl orthosilicate and ammonia, and stir the reaction under light-proof and oxygen-free conditions. After centrifugation, elution and drying, the nitrogen and phosphorus co-doped carbon dots@ZIF-67@MIPs composite material is obtained.

[0011] Further improvements were made to the preparation method of nitrogen and phosphorus co-doped carbon dots @ZIF-67@MIPs composite materials: Preferably, in step S3, the concentration of ammonia is 25-28 wt%, and the mass ratio of ZIF-67 in the ZIF-67 dispersion to N,P-CDs, sulfadiazine, 3-aminopropyltriethoxysilane, tetraethyl orthosilicate and ammonia in the N,P-CDs solution is 1:(0.0022-0.022):(2-10):(15-32):(30-80):(5-10).

[0012] Preferably, the stirring reaction time in step S3 is 8-12 h. After centrifugation to collect the product, the template molecules are removed by elution with a methanol / acetic acid mixed solution, wherein the volume ratio of methanol to acetic acid in the methanol / acetic acid mixed solution is 9:1.

[0013] Preferably, the preparation steps of the N,P-CDs solution in step S1 are as follows: 0.2-0.8 g of citric acid, 6-12 mL of ethylenediamine and 2-8 mL of phosphoric acid are added sequentially to 20 mL of deionized water, mixed evenly, and then transferred to a reaction vessel. The mixture is subjected to hydrothermal reaction at 160-200 °C for 6-10 h. The solution is then purified by dialysis using a dialysis bag with a molecular weight cutoff of 500-1000 Da for 12-48 h to obtain an N,P-CDs solution with a concentration of 0.1-1 mg / mL.

[0014] Preferably, the preparation steps of the ZIF-67 dispersion in step S2 are as follows: weigh cobalt nitrate hexahydrate and 2-methylimidazole in a molar ratio of 1:(2-32) and dissolve them separately in methanol; wherein the cobalt ion concentration in the methanol solution of cobalt nitrate hexahydrate is 45-100 mM; and the concentration of 2-methylimidazole in the methanol solution of 2-methylimidazole is 200-300 mM; mix the two solutions and stir at a rate of 200-600 r / min at a temperature of 22-30 ℃ for 8-12 h; the product is centrifuged, washed, dried, and dispersed in methanol to obtain a ZIF-67 dispersion with a concentration of 0.5-2 mg / mL.

[0015] The third objective of this invention is to provide an application of the above-mentioned nitrogen and phosphorus co-doped carbon dot @ZIF-67@MIPs composite material in the detection of sulfadiazine. The composite material is mixed with the sample to be tested, and a fluorescence spectrophotometer is used with an excitation wavelength of 360 nm. The concentration of sulfadiazine in the actual sample is quantitatively detected based on the standard curve and the change in fluorescence intensity.

[0016] The advantages of this invention compared to the prior art are as follows: (1) This invention provides an N,P-CDs@ZIF-67@MIPs composite material. This composite fluorescent polymer uses N,P-CDs as a fluorescence source and anchors N,P-CDs onto the ZIF-67 support through a low loading method. The benefits of low loading are: ① avoiding fluorescence self-quenching caused by the aggregation of N,P-CDs on the ZIF-67 surface; ② reducing steric hindrance and providing a smooth active surface for the uniform coating of the subsequent MIPs shell; ③ saving carbon dots and reducing costs; ④ keeping the ZIF-67 pores open and playing its role in pre-enriching the target material. Regarding the doping mechanism of N,P-CDs themselves: N and P form a "push-pull" electronic system in the carbon skeleton - the electron-donating effect of P expands π conjugation and suppresses carrier recombination, and the electron-withdrawing effect of N forms a local electron enrichment region. The two work together to reduce non-radiative transitions and optimize the surface state (P replaces edge oxygen atoms to repair the conjugated structure, and N introduces hydrophilic groups such as amino groups). Compared to single N doping, N,P co-doping improves fluorescence quantum yield, photochemical stability, and water solubility. ZIF-67, as a support, protects the fluorescence properties of N,P-CDs and provides support for the growth of MIPs. MIPs, as the outer layer, have specific recognition capabilities for sulfadiazine, effectively eliminating the influence of interfering substances. The synergistic effect of these three factors (low-load anchoring + ZIF-67 protection and enrichment + specific recognition by MIPs) gives this polymer material the advantages of high specificity, high stability, and high accuracy.

[0017] (2) This invention uses a surface sol-gel method to prepare N,P-CDs@ZIF-67@MIPs composite materials. Nitrogen and phosphorus-doped carbon dots are prepared using a hydrothermal method, and cobalt-based metal-organic framework ZIF-67 is synthesized at room temperature. Then, N,P-CDs and ZIF-67 are mixed at low concentrations, achieving low-load anchoring through physical adsorption. Using sol-gel surface imprinting technology, sulfadiazine is used as a template, and APTES and TEOS are added to polymerize on the surface of N,P-CDs@ZIF-67 to obtain the molecularly imprinted composite material. This method is simple to operate, has mild conditions, and produces uniform coating. It can achieve precise coating of the ZIF-67 intermediate layer and the MIPs outer layer on the surface of N,P-CDs, effectively avoiding problems such as uneven coating and weak bonding in traditional coating methods. It significantly improves the stability and fluorescence performance of the material, and has good repeatability, making it easy to scale up production.

[0018] (3) The N,P-CDs@ZIF-67@MIPs composite material of the present invention provides a new technical means for the rapid and accurate detection of sulfadiazine, and has broad application prospects. The detection method can be used to detect sulfadiazine in actual samples (aquatic environment, milk). The pretreatment method is simple and the results are reliable. The recovery rate of lake water samples is between 95.35% and 103.49%, and the recovery rate of milk samples is between 87.04% and 106.70%, with relative standard deviations of <5.10%. The detection method is simple to operate, has a fast response, a short incubation time, does not require complex instruments and equipment, can realize rapid on-site detection, has low detection cost, and is suitable for rapid screening of large batches of samples. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the preparation process of the nitrogen and phosphorus co-doped carbon dot @ZIF-67@MIPs composite material of the present invention.

[0020] Figure 2 In the middle, (A) and (B) are TEM images of N,P-CDs prepared in Preparation Example 1 and N,P-CDs@ZIF-67@MIPs prepared in Example 1, respectively; (C), (D), and (E) are SEM images of ZIF-67 prepared in Preparation Example 2, N,P-CDs@ZIF-67@MIPs prepared in Example 1, and N,P-CDs@ZIF-67@NIPs prepared in Comparative Example 1, respectively.

[0021] Figure 3 Infrared spectra of ZIF-67 prepared in Example 2 and N,P-CDs@ZIF-67@MIPs prepared in Example 1.

[0022] Figure 4 X-ray diffraction patterns of ZIF-67 in Example 2, N,P-CDs@ZIF-67@MIPs in Example 1, and N,P-CDs@ZIF-67@NIPs in Comparative Example 1 were prepared.

[0023] Figure 5 This is a stability graph of the fluorescence intensity of N,P-CDs@ZIF-67@MIPs in Example 1.

[0024] Figure 6 The graph shows the linear relationship between the fluorescence intensity of N,P-CDs@ZIF-67@MIPs in Example 1 and N,P-CDs@ZIF-67@NIPs in Comparative Example 1 and the response time of sulfadiazine.

[0025] Figure 7 This is a graph showing the changes in the imprinting factor of N,P-CDs@ZIF-67@MIPs after binding with sulfadiazine at different pH values ​​in Example 1.

[0026] Figure 8 The figure shows the relationship between the fluorescence intensity of N,P-CDs@ZIF-67@MIPs and the concentration of sulfadiazine in Example 1, where (A) is the variation curve and (B) is the linear relationship curve.

[0027] Figure 9 This is a selectivity diagram of N,P-CDs@ZIF-67@MIPs for sulfadiazine and its analogues under the same concentration conditions in Example 1.

[0028] Figure 10 The image shows the anti-interference recognition performance of N,P-CDs@ZIF-67@MIPs against the target substance sulfadiazine (SDZ) in Example 1 (a binary coexistence system in which SDZ is mixed with STZ and TC in proportion). Detailed Implementation

[0029] 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.

[0030] Preparation Example 1 This preparation example provides a method for preparing an N,P-CDs solution, comprising the following steps: S1. Add 0.6 g citric acid, 10 mL ethylenediamine and 5 mL phosphoric acid to 20 mL deionized water in sequence, mix well and transfer to a polytetrafluoroethylene-lined reactor. After sealing, perform hydrothermal reaction at 180 °C for 8 h. After the reaction is completed, allow it to cool naturally to room temperature. S2. Centrifuge at 8000 r / min for 15 min, collect the supernatant, filter through a 0.22 μm filter membrane, and then dialyze through a dialysis bag (molecular weight cutoff 500 Da) for 48 h to obtain a N,P-CDs solution with a concentration of 0.6 mg / mL, and store at 4 ℃ for later use.

[0031] Electron microscopy revealed that the particle size of the nitrogen- and phosphorus co-doped carbon dots (N,P-CDs) was 2-4 nm.

[0032] Preparation Example 2 This preparation example provides a method for preparing ZIF-67, including the following steps: S1. Weigh 3 mmol / L of cobalt nitrate hexahydrate and 12 mmol / L of 2-methylimidazole (molar ratio 1:4), and dissolve them separately in methanol; wherein the concentration of cobalt ion in the methanol solution of cobalt nitrate hexahydrate is 60 mM; and the concentration of 2-methylimidazole in the methanol solution of 2-methylimidazole is 240 mM. S2. Mix the two solutions and stir at 400 r / min at 25 ℃ for 12 h. Collect the purple precipitate by centrifugation, wash with methanol 5 times to remove unreacted raw materials, and dry under vacuum to obtain ZIF-67 powder. Disperse the ZIF-67 powder in methanol and sonicate for 30 min to obtain a ZIF-67 dispersion with a concentration of 1.125 mg / mL.

[0033] Electron microscopy revealed that the prepared ZIF-67 exhibited a rhombic dodecahedral crystal structure with a particle size of 450 nm.

[0034] Preparation Example 3 This preparation example provides a method for preparing an N-CDs solution. The specific steps are the same as in Preparation Example 1, except that phosphoric acid is not added in step S1. Nitrogen-doped carbon dot N-CDs are finally obtained.

[0035] Electron microscopy revealed that the nitrogen-doped carbon dots (N-CDs) had a particle size of 2–4 nm.

[0036] Preparation Example 4 This preparation example provides a method for preparing ZIF-8, including the following steps: S1. Weigh 5 mmol of zinc nitrate hexahydrate and 40 mmol of 2-methylimidazole, dissolve them in methanol respectively, and prepare metal salt solution and ligand solution respectively.

[0037] S2. Mix the two solutions above and stir continuously at room temperature for 1 h. Centrifuge to collect the white precipitate, wash with methanol 5 times to remove unreacted raw materials, and vacuum dry to obtain ZIF-8 powder.

[0038] Electron microscopy revealed that the ZIF-8 powder had a particle size of 100-900 nm.

[0039] Example 1 This embodiment provides a method for preparing N,P-CDs@ZIF-67@MIPs, the process is as follows: Figure 1 As shown, it includes the following steps: S1. Take 40 mL of the ZIF-67 dispersion (containing 45 mg of ZIF-67) prepared in Preparation Example 2, add 600 μL of the N,P-CDs dispersion (containing 0.36 mg of N,P-CDs) prepared in Preparation Example 1, and stir thoroughly to obtain the N,P-CDs@ZIF-67 precursor mixture.

[0040] S2. Transfer the precursor mixture into a three-necked flask and add 255 mg of sulfadiazine (SDZ, template molecule), 1076 mg of 3-aminopropyltriethoxysilane (APTES), 1350 mg of tetraethyl orthosilicate (TEOS), and 364 mg of ammonia solution (25 wt%) in sequence.

[0041] S3. The reaction was continuously stirred for 10 h under completely dark and oxygen-free conditions. The product was collected by centrifugation at 8000 r / min. The template molecules were removed by elution with a methanol / acetic acid mixed solution (volume ratio 9:1). After washing, the product was vacuum dried to obtain nitrogen and phosphorus co-doped carbon dots @ZIF-67@MIPs composite material, denoted as N,P-CDs@ZIF-67@MIPs, which was stored under cold storage for later use.

[0042] Example 2 This embodiment provides a method for preparing N,P-CDs@ZIF-67@MIPs. The specific steps are the same as in Example 1, except that 675 mg of 3-aminopropyltriethoxysilane (APTES) is added in step S2.

[0043] Example 3 This embodiment provides a method for preparing N,P-CDs@ZIF-67@MIPs. The specific steps are the same as in Example 1, except that 1440 mg of 3-aminopropyltriethoxysilane (APTES) is added in step S2.

[0044] Example 4 This embodiment provides a method for preparing N,P-CDs@ZIF-67@MIPs. The specific steps are the same as in Example 1, except that 3600 mg of tetraethyl orthosilicate (TEOS) is added in step S2.

[0045] Example 5 This embodiment provides a method for preparing N,P-CDs@ZIF-67@MIPs, including the following steps: S1. Add 0.2 g citric acid, 6 mL ethylenediamine and 2 mL phosphoric acid sequentially to 20 mL deionized water, mix well and transfer to a polytetrafluoroethylene-lined reactor. Seal and hydrothermally react at 160 ℃ for 6 h. After the reaction is complete, allow to cool naturally to room temperature. Centrifuge at 8000 r / min for 15 min, collect the supernatant, filter through a 0.22 μm filter membrane, and then dialyze through a dialysis bag with a molecular weight cutoff of 500 Da for 12 h to obtain a N,P-CDs solution with a concentration of 0.1 mg / mL. Store at 4 ℃ for later use.

[0046] S2. Weigh 3 mmol of cobalt nitrate hexahydrate and 6 mmol of 2-methylimidazole (molar ratio 1:2), and dissolve them separately in methanol; the cobalt ion concentration in the methanol solution of cobalt nitrate hexahydrate is 45 mM; the 2-methylimidazole concentration in the methanol solution of 2-methylimidazole is 200 mM. Mix the two solutions and stir at 200 r / min at 22 ℃ for 8 h. Centrifuge to collect the purple precipitate, wash it 5 times with methanol, and vacuum dry to obtain ZIF-67 powder. Disperse the ZIF-67 powder in methanol and sonicate for 30 min to obtain a ZIF-67 dispersion with a concentration of 0.5 mg / mL.

[0047] S3. Take 90 mL of the above ZIF-67 dispersion (containing 45 mg ZIF-67), add 990 μL of the above N,P-CDs solution (containing 0.099 mg N,P-CDs), and stir thoroughly. Transfer to a three-necked flask, and add 90 mg sulfadiazine (SDZ, template molecule), 675 mg 3-aminopropyltriethoxysilane (APTES), 1350 mg tetraethyl orthosilicate (TEOS), and 225 mg ammonia solution (concentration 25 wt%) in sequence. Under completely dark and anaerobic conditions, continue stirring for 8 h. Collect the product by centrifugation at 8000 r / min, and remove the template molecule by elution with a methanol / acetic acid mixture (volume ratio 9:1). After washing, vacuum dry to obtain nitrogen and phosphorus co-doped carbon dots@ZIF-67@MIPs composite material.

[0048] Characterization showed that the composite material prepared in this embodiment has an overall irregular spherical structure with a particle size of approximately 200-400 nm; the N,P-CDs have a particle size of 2-4 nm; and the molecularly imprinted polymer layer has a thickness of approximately 12 nm.

[0049] Example 6 This embodiment provides a method for preparing N,P-CDs@ZIF-67@MIPs, including the following steps: S1. Add 0.8 g citric acid, 12 mL ethylenediamine and 8 mL phosphoric acid sequentially to 20 mL deionized water, mix well and transfer to a polytetrafluoroethylene-lined reactor. Seal and hydrothermally react at 200℃ for 10 h. After the reaction is complete, allow to cool naturally to room temperature. Centrifuge at 8000 r / min for 15 min, collect the supernatant, filter through a 0.22 μm filter membrane, and then dialyze through a dialysis bag with a molecular weight cutoff of 1000 Da for 48 h to obtain a N,P-CDs solution with a concentration of 1.0 mg / mL. Store at 4 ℃ for later use.

[0050] S2. Weigh 3 mmol of cobalt nitrate hexahydrate and 96 mmol of 2-methylimidazole (molar ratio 1:32), and dissolve them separately in methanol; the cobalt ion concentration in the methanol solution of cobalt nitrate hexahydrate is 100 mM; the 2-methylimidazole concentration in the methanol solution of 2-methylimidazole is 300 mM. Mix the two solutions and stir at 600 r / min at 30 ℃ for 12 h. Collect the purple precipitate by centrifugation, wash it 5 times with methanol, and dry it under vacuum to obtain ZIF-67 powder. Disperse the ZIF-67 powder in methanol and sonicate for 30 min to obtain a ZIF-67 dispersion with a concentration of 2.0 mg / mL.

[0051] S3. Take 22.5 mL of the above ZIF-67 dispersion (containing 45 mg ZIF-67), add 990 μL of the above N,P-CDs solution (containing 0.99 mg N,P-CDs), and stir thoroughly. Transfer to a three-necked flask, and add 450 mg sulfadiazine (SDZ, template molecule), 1440 mg 3-aminopropyltriethoxysilane (APTES), 3600 mg tetraethyl orthosilicate (TEOS), and 450 mg ammonia solution (concentration 28 wt%) in sequence. Under completely dark and anaerobic conditions, continue stirring for 12 h. Collect the product by centrifugation at 8000 r / min, and remove the template molecule by elution with a methanol / acetic acid mixture (volume ratio 9:1). After washing, vacuum dry to obtain nitrogen and phosphorus co-doped carbon dots@ZIF-67@MIPs composite material.

[0052] Characterization showed that the composite material prepared in this embodiment has an overall irregular spherical structure with a particle size of about 600-1000 nm; the N,P-CDs particle size is 2-4 nm; and the thickness of the molecularly imprinted polymer layer is about 38 nm.

[0053] Comparative Example 1 This comparative example provides a method for preparing N,P-CDs@ZIF-67@NIPs. The specific steps are the same as in Example 1, except that sulfadiazine (SDZ, template molecule) is not added in step S2. N,P-CDs@ZIF-67@NIPs are finally obtained.

[0054] Comparative Example 2 This comparative example provides a method for preparing N-CDs@ZIF-67@MIPs. The specific steps are the same as in Example 1, except that N single-doped carbon dots (N-CDs) from Preparation Example 3 are used instead of N,P co-doped carbon dots (N,P-CDs) to finally obtain N-CDs@ZIF-67@MIPs.

[0055] Comparative Example 3 This comparative example provides a method for preparing N,P-CDs@ZIF-8@MIPs. The specific steps are the same as in Example 1, except that ZIF-8 from Preparation Example 4 is used instead of ZIF-67. N,P-CDs@ZIF-8@MIPs are finally obtained.

[0056] Comparative Example 4 This comparative example provides a method for preparing N-CDs@ZIF-8@MIPs. The specific steps are the same as in Example 1, except that N-doped carbon dots (N-CDs) from Preparation Example 3 are used instead of N,P co-doped carbon dots (N,P-CDs), and ZIF-8 from Preparation Example 4 is used instead of ZIF-67. Finally, N-CDs@ZIF-8@MIPs are obtained.

[0057] Figure 1 The flowchart below shows the N,P-CDs@ZIF-67@MIPs polymer prepared according to the present invention. In this invention, N,P-CDs and ZIF-67 are synthesized sequentially. The two are then physically adsorbed to load N,P-CDs onto ZIF-67. Subsequently, template molecules sulfadiazine, APTES, TEOS, and ammonia are added to carry out molecularly imprinted polymerization. Finally, the template molecules are eluted to remove them, yielding the N,P-CDs@ZIF-67@MIPs fluorescent polymer.

[0058] Performance testing 1. Characterization by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) The N,P-CDs prepared in Preparation Example 1 and the N,P-CDs@ZIF-67@MIPs prepared in Example 1 were subjected to TEM testing, and the results are as follows. Figure 2 (A) and Figure 2 As shown in (B). Figure 2 (A) shows that the size of N,P-CDs particles is 2-4 nm; Figure 2 (B) shows that the N,P-CDs@ZIF-67@MIPs have a near-spherical structure with an imprinted layer on its surface, which is 10-40 nm thick.

[0059] The ZIF-67 prepared in Preparation Example 2, the N,P-CDs@ZIF-67@MIPs prepared in Example 1, and the N,P-CDs@ZIF-67@NIPs prepared in Comparative Example 1 were observed by SEM. The test results are as follows. Figure 2 As shown in (C), 2(D) and 2(E). Figure 2 (C) shows that pure ZIF-67 exhibits a regular rhombic dodecahedral crystal structure with a grain size of 100-900 nm. The crystal surface is smooth with clear edges and corners, without obvious agglomeration, and has high crystallinity, which is consistent with the morphological characteristics of typical ZIF-67 (MOF) materials. Figure 2(D) is the SEM image of N,P-CDs@ZIF-67@MIPs. Figure 2 (E) is a SEM image of N,P-CDs@ZIF-67@NIPs. The comparison shows that N,P-CDs@ZIF-67@MIPs still retains the core crystal framework of ZIF-67, but the crystal surface becomes rough and is covered with a uniform amorphous polymer layer, and the particle size is slightly increased.

[0060] 2. Fourier Transform Infrared Spectroscopy Characterization (FT-IR) Infrared spectroscopy was performed on ZIF-67 prepared in Example 2 and N,P-CDs@ZIF-67@MIPs prepared in Example 1. The results are as follows: Figure 3 As shown. Figure 3 Curve A in the middle is the infrared spectrum of ZIF-67, at 428 cm⁻¹. -1 The absorption peak appearing at this point is attributed to the stretching vibration of the Co-N bond, a characteristic peak of the ZIF-67 metal-organic framework, proving that Co... 2+ Successful coordination with the imidazole ligand. Curve B is the infrared spectrum of N,P-CDs@ZIF-67@MIPs, retaining the core characteristic peaks of ZIF-67, indicating that the ZIF-67 framework was not destroyed after recombination. Meanwhile, curve B shows a peak at 1083 cm⁻¹. -1 An absorption peak at 1638 cm⁻¹ appeared, attributed to the Si-O-Si asymmetric stretching vibration. This is direct evidence of the molecularly imprinted polymer layer formed by the hydrolytic condensation of APTES and TEOS, and is a characteristic peak of molecularly imprinted polymers. This peak is not present in the infrared spectrum of pure ZIF-67. -1 The absorption peak at 2920 cm⁻¹ is attributed to the in-plane shear vibration of -NH₂, originating from the amino groups on the surface of N,P-CDs and the terminal amino groups of APTES, confirming that N,P-CDs were successfully loaded and that APTES participated in the polymerization. -1 The absorption peaks appearing at this point are attributed to the asymmetric stretching vibration of -CH2-, originating from the alkyl chains in APTES / TEOS and the alkyl groups on the surface of N,P-CDs, further confirming the formation of the molecularly imprinted layer.

[0061] 3. X-ray diffraction characterization (XRD) XRD tests were performed on the ZIF-67 prepared in Preparation Example 2, the N,P-CDs@ZIF-67@MIPs in Example 1, and the N,P-CDs@ZIF-67@NIPs in Comparative Example 1. The results are as follows: Figure 4 As shown, Figure 4In the diagram, curve (A) shows the XRD pattern of ZIF-67, whose diffraction peak positions are consistent with the ZIF-67 standard card data, indicating that a pure phase ZIF-67 with complete crystal structure and ordered structure has been obtained. Curves (B) and (C) are the XRD patterns of N,P-CDs@ZIF-67@MIPs and N,P-CDs@ZIF-67@NIPs, respectively. The characteristic diffraction peaks of ZIF-67 are still present in the figures, but the intensity has decreased significantly and the peak shape has broadened. The original sharp peaks have become shorter and blunter, indicating that the crystal structure is still there, but it is "masked" by the amorphous phase. A broadened diffuse diffraction peak appears at around 21°, which is mainly due to the joint contribution of polymer MIPs and N,P-CDs, indicating that MIPs have been successfully coated on the material surface. NIPs have also been successfully coated, and no obvious crystal structure difference between them and MIPs was observed in the XRD pattern. The above results indirectly confirm the successful synthesis of N,P-CDs@ZIF-67@MIPs and N,P-CDs@ZIF-67@NIPs composite materials.

[0062] 4. Measure fluorescence intensity N,P-CDs@ZIF-67@MIPs prepared with different ratios of APTES and TEOS in Examples 1, 2, 3, and 4, and N,P-CDs@ZIF-67@NIPs prepared in Comparative Example 1, were dispersed in 5 μg / mL SDZ standard solution, respectively. The fluorescence intensity was measured using a fluorescence spectrophotometer, and the imprinting factor was calculated using the following formula: IF=[(F0 / F)-1] MIPs / [(F0 / F)-1] NIPs Among them, when the quantitative relationship of template molecules, functional monomers and crosslinking agents in Example 1 is adopted, the imprinting factor is the largest of 2.26.

[0063] 5. Linear relationship between relative fluorescence intensity and sulfadiazine concentration Under the same conditions, the detection performance of the composite materials prepared in Example 1 and Comparative Examples 2-4 as sensing materials for fluorescence sensors was measured in the linear range of 1-10 μg / mL for the target molecule sulfadiazine. The results show that: The linear relationship between the relative fluorescence intensity of N,P-CDs@ZIF-67@MIPs in Example 1 and the concentration of sulfadiazine was y = 0.0857C + 0.9779, with a detection limit of 0.045 μg / mL and an imprinting factor of 2.26.

[0064] The linear relationship between the relative fluorescence intensity of N-CDs@ZIF-67@MIPs obtained in Comparative Example 2 and the concentration of sulfadiazine was y=0.04229C+1.08381, with a detection limit of 0.101 μg / mL and an imprinting factor of 1.63.

[0065] The linear relationship between the relative fluorescence intensity of N,P-CDs@ZIF-8@MIPs obtained in Comparative Example 3 and the concentration of sulfadiazine was y = 0.03868C + 1.08932, with a detection limit of 0.131 μg / mL and an imprinting factor of 1.58.

[0066] The linear relationship between the relative fluorescence intensity of N-CDs@ZIF-8@MIPs obtained in Comparative Example 4 and the concentration of sulfadiazine was y = 0.03437C + 1.00733, with a detection limit of 0.249 μg / mL and an imprinting factor of 1.54.

[0067] The data above show that the N,P co-doped carbon dots and ZIF-67 exhibit a synergistic enhancement effect.

[0068] 6. Fluorescence performance study Weigh out the N,P-CDs@ZIF-67@NIPs polymer powder of Comparative Example 1 and the N,P-CDs@ZIF-67@MIPs polymer powder of Example 1, add deionized water to prepare a suspension of 1 mg / mL, and store at 4 ℃ in the dark for later use.

[0069] (1) Stability test: Take a clean 5 mL centrifuge tube, add 1 mL of the above N,P-CDs@ZIF-67@MIPs suspension, then add PBS buffer at pH = 7.0, and mix thoroughly. Transfer the mixed suspension to the sample detection cell of the fluorescence spectrophotometer. Measure the fluorescence intensity every 5 min within 1 hour, record the changes in fluorescence intensity at different time points, and take the average value for each sample in three tests.

[0070] Depend on Figure 5 As can be seen, within the 1-hour testing period, the fluorescence intensity of N,P-CDs@ZIF-67@MIPs remained within a relatively stable range, without any obvious upward or downward trend, and the intensity fluctuation was controlled within a small range. This phenomenon fully demonstrates that N,P-CDs@ZIF-67@MIPs possesses good fluorescence stability, and its luminescence performance is not easily affected by short-term external environmental disturbances.

[0071] The composite materials prepared in Examples 2-6 were tested under the same conditions. The test results confirmed that they could effectively detect sulfadiazine, and their fluorescence quenching behavior was basically the same as that in Example 1.

[0072] (2) Response time experiment: Take 1 mL of the above-mentioned N,P-CDs@ZIF-67@MIPs suspension and N,P-CDs@ZIF-67@NIPs suspension respectively, add SDZ standard solution and PBS buffer at pH = 7.0, shake at room temperature, and measure the fluorescence intensity every 5 min within 45 min. The results are as follows. Figure 6 As shown, during the rapid response phase of 0-30 min, the fluorescence intensity ratio (F0 / F) of the MIPs sample increased at a significantly faster rate than that of the NIPs sample. This difference is attributed to the formation of specific imprinted cavities on the surface of the MIPs that match the size and shape of the target molecule, enabling rapid recognition and binding; while NIPs rely solely on non-specific interactions. After 30 min, both curves tended to plateau, reaching response equilibrium. This indicates that the response rate and relative fluorescence intensity of MIPs to sulfadiazine are higher than those of NIPs, demonstrating specific recognition capabilities.

[0073] (3) Effect of pH on fluorescence intensity: Take 1 mL of sulfadiazine standard solution with a concentration of 3 μg / mL, add 1 mL of N,P-CDs@ZIF-67@MIPs suspension, and adjust the volume to 5 mL with buffer solutions of pH 3, 5, 7, 9 and 11 respectively; place the prepared solution at room temperature, and after shaking and incubating for 30 min, measure the change in fluorescence intensity of the system under different pH conditions.

[0074] Figure 7 The graphs show the imprinting factor (IF) of N,P-CDs@ZIF-67@MIPs as a function of solution pH, examining the material's pH response characteristics. Within the pH range of 3-7, the imprinting factor increases rapidly with increasing pH, reaching its maximum at pH 7, which is the optimal pH for the material's response. Within the pH range of 7-11, the imprinting factor decreases slowly with increasing pH, but still remains at a relatively high level. This indicates that the material is suitable for use in neutral or weakly alkaline environments.

[0075] The composite materials prepared in Examples 2-6 were tested under the same conditions, and the results confirmed that the composite materials prepared in Examples 2-6 are also suitable for use in neutral or weakly alkaline environments.

[0076] (4) Linear relationship between fluorescence intensity and SDZ concentration: Sulfadiazine SDZ standard solutions with concentrations of 1 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL and 10 μg / mL were prepared. 1 mL of the standard solution was mixed with 1 mL of the above N,P-CDs@ZIF-67@MIPs suspension and incubated at 25 ℃ and pH 7.0 for 30 min. A fluorescence spectrophotometer was used, with an excitation wavelength of 360 nm and an emission range of 380-600 nm. The fluorescence intensity was measured by fitting the sulfadiazine concentration on the x-axis and F0 / F (F is the fluorescence intensity of the standard solution system and F0 is the fluorescence intensity of the blank system) on the y-axis to obtain a standard curve.

[0077] Figure 8In Figure (A), the fluorescence intensity of N,P-CDs@ZIF-67@MIPs in Example 1 changes with the concentration of sulfadiazine. Figure 8 (B) shows the linear relationship between the relative fluorescence intensity of N,P-CDs@ZIF-67@MIPs and the concentration of sulfadiazine, with the regression equation being y = 0.0857C + 0.9779, R0. 2 = 0.9964; detection limit is 0.045 μg / mL.

[0078] (5) Selectivity Experiment: Sulfamethazine (SDZ), sulfacetamide (SA), sulfadiazine (SM2), sulfathiazole (STZ), and sulfapyridine (SP), which have similar structures, were prepared into solutions of equal concentration. Then, 1 mL of MIPs and NIPs solutions were added to equal volumes of sulfadiazine and similar solutions, respectively, and the fluorescence signal intensity of the polymers before and after adsorption was detected using a fluorescence spectrophotometer. Based on the changes in fluorescence intensity, the differences in the selectivity of molecularly imprinted sites for recognizing sulfadiazine and structurally similar substances were compared and analyzed.

[0079] Figure 9 The figure shows the selectivity of N,P-CDs@ZIF-67@MIPs for sulfadiazine and its analogues under the same concentration conditions. As can be seen from the figure, the fluorescence response of N,P-CDs@ZIF-67@MIPs to sulfadiazine is significantly higher than that of the other four sulfonamide antibiotics. This result is attributed to the formation of specific imprinted cavities on the surface of the MIPs that are highly complementary to the spatial structure, size, and functional groups of SDZ, enabling them to target and recognize SDZ, thus exhibiting specific selectivity for sulfadiazine.

[0080] (6) Anti-interference experiment: Sulfathiazole (STZ) and tetracycline (TC) were selected as competing substrates, and five mixed test solutions with different concentration ratios were prepared (SDZ:STZ=1:0, 1:1, 1:2; SDZ:TC=1:1, 1:2). The test solution was added to 1 mL of MIPs and NIPs solutions respectively, and the fluorescence signal intensity of the polymer before and after adsorption was measured. This was used to compare and analyze the specific recognition of template molecules and anti-interference ability of molecular imprinted sites.

[0081] Figure 10This image shows the anti-interference recognition performance of N,P-CDs@ZIF-67@MIPs for the target substance sulfadiazine (SDZ) in Example 1 (a binary coexistence system of SDZ mixed with STZ and TC in different proportions). The image shows the binary system coexisting with sulfadiazine. As can be seen from the image, even under interference from high concentrations of the structural analog STZ and the non-structural analog TC, the fluorescence response of SDZ shows no significant fluctuation, and the intensity change is negligible. This confirms that the specific imprinted pores on the surface of the MIPs are highly complementary to SDZ in terms of spatial configuration and functional group arrangement, enabling precise targeting and recognition of SDZ unaffected by interfering substances. This sensing system exhibits good anti-interference performance and is suitable for the quantitative detection of SDZ in complex matrices.

[0082] 8. Analysis of actual samples Lake water samples: After standing overnight, the samples were filtered through a 0.22 μm filter membrane, and SDZ standard solution was added. The solutions were then diluted with PBS buffer to concentrations of 2 μg / mL, 5 μg / mL, and 8 μg / mL. The N,P-CDs@ZIF-67@MIPs suspension was mixed with the lake water sample solution and incubated at room temperature for 30 min. Fluorescence intensity was measured, with each concentration measured three times. The recovery rate and standard deviation were calculated. The results are shown in Table 1. The recovery rate ranged from 95.35% to 103.49%, and the relative standard deviation was <5.10%.

[0083] Table 1 Recovery rates of N,P-CDs@ZIF-67@MIPs in lake water

[0084] Milk samples: After removing fat and precipitated protein, the samples were centrifuged and filtered through a 0.22 μm filter membrane. SDZ standard solution was added, and the solutions were diluted with PBS buffer (pH 7.0) to concentrations of 2 μg / mL, 5 μg / mL, and 8 μg / mL. The N,P-CDs@ZIF-67@MIPs suspension was mixed with the milk sample solution and incubated at room temperature for 30 min. Fluorescence intensity was measured, with each concentration measured three times. The recovery rate and standard deviation were calculated. The results are shown in Table 2. The recovery rate ranged from 87.04% to 106.70%, and the relative standard deviation was <5.10%.

[0085] Table 2. Recovery rates of N,P-CDs@ZIF-67 @MIPs in milk samples

[0086] As shown in Tables 1 and 2, N,P-CDs@ZIF-67@MIPs exhibit good recoveries within the linear range, ranging from 95.35% to 103.49% for lake water and from 87.04% to 106.70% for milk samples, with relative standard deviations of <5.10% for both. The N,P-CDs@ZIF-67@MIPs composite material demonstrates good accuracy and precision in both matrices, meeting the requirements for practical sample testing.

[0087] 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 nitrogen- and phosphorus co-doped carbon dot@ZIF-67@MIPs composite material, characterized in that, The composite material is composed of nitrogen and phosphorus co-doped carbon dots @ zeolite imidazole ester framework-67 @ molecularly imprinted polymer, with an overall irregular spherical structure and a particle size of 200-1000 nm. The polymer uses a cobalt-based metal-organic framework ZIF-67 as a carrier, and the surface of the carrier is loaded with nitrogen and phosphorus co-doped carbon dots. The surface of the carrier and the nitrogen and phosphorus co-doped carbon dots is coated with a molecularly imprinted polymer layer, which has sulfadiazine recognition sites.

2. The nitrogen and phosphorus co-doped carbon dot@ZIF-67@MIPs composite material according to claim 1, characterized in that, The ZIF-67 has a rhombic dodecahedral crystal structure with a particle size of 100-900 nm.

3. The nitrogen and phosphorus co-doped carbon dot@ZIF-67@MIPs composite material according to claim 1, characterized in that, The nitrogen and phosphorus co-doped carbon dots have a particle size of 2-4 nm and a mass ratio of (0.0022-0.022):1 with the carrier ZIF-67.

4. The nitrogen and phosphorus co-doped carbon dot @ZIF-67@MIPs composite material according to claim 1 or 3, characterized in that, The thickness of the molecularly imprinted polymer layer is 10-40 nm.

5. A method for preparing the nitrogen and phosphorus co-doped carbon dot @ZIF-67@MIPs composite material according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Prepare a nitrogen-phosphorus co-doped carbon dot solution, i.e., N,P-CDs solution, by hydrothermal method using citric acid, ethylenediamine and phosphoric acid. S2. ZIF-67 powder was prepared by cobalt nitrate hexahydrate and 2-methylimidazole through a coordination reaction, and then dispersed in a solvent to obtain ZIF-67 dispersion. S3. Mix the N,P-CDs solution with the ZIF-67 dispersion evenly, add the template molecules sulfadiazine, 3-aminopropyltriethoxysilane, tetraethyl orthosilicate and ammonia, and stir the reaction under light-proof and oxygen-free conditions. After centrifugation, elution and drying, the nitrogen and phosphorus co-doped carbon dots@ZIF-67@MIPs composite material is obtained.

6. The method for preparing the nitrogen and phosphorus co-doped carbon dot @ZIF-67@MIPs composite material according to claim 5, characterized in that, In step S3, the concentration of ammonia is 25-28 wt%, and the mass ratio of ZIF-67 in the ZIF-67 dispersion to N,P-CDs, sulfadiazine, 3-aminopropyltriethoxysilane, tetraethyl orthosilicate and ammonia in the N,P-CDs solution is 1:(0.0022-0.022):(2-10):(15-32):(30-80):(5-10).

7. The method for preparing the nitrogen and phosphorus co-doped carbon dot@ZIF-67@MIPs composite material according to claim 5 or 6, characterized in that, In step S3, the stirring reaction time is 8-12 h. After centrifugation to collect the product, the template molecules are removed by elution with a methanol / acetic acid mixed solution. The volume ratio of methanol to acetic acid in the methanol / acetic acid mixed solution is 9:

1.

8. The method for preparing the nitrogen and phosphorus co-doped carbon dot@ZIF-67@MIPs composite material according to claim 5, characterized in that, The preparation steps of the N,P-CDs solution in step S1 are as follows: 0.2-0.8 g of citric acid, 6-12 mL of ethylenediamine and 2-8 mL of phosphoric acid are added sequentially to 20 mL of deionized water, mixed evenly and then transferred to a reaction vessel. The mixture is then subjected to hydrothermal reaction at 160-200 °C for 6-10 h. The solution is purified by dialysis using a dialysis bag with a molecular weight cutoff of 500-1000 Da for 12-48 h to obtain an N,P-CDs solution with a concentration of 0.1-1 mg / mL.

9. The method for preparing the nitrogen and phosphorus co-doped carbon dot@ZIF-67@MIPs composite material according to claim 5, characterized in that, The preparation steps of the ZIF-67 dispersion in step S2 are as follows: Weigh cobalt nitrate hexahydrate and 2-methylimidazole in a molar ratio of 1:(2-32) and dissolve them separately in methanol; wherein the cobalt ion concentration in the methanol solution of cobalt nitrate hexahydrate is 45-100 mM; and the concentration of 2-methylimidazole in the methanol solution of 2-methylimidazole is 200-300 mM; mix the two solutions and stir at a rate of 200-600 r / min at a temperature of 22-30 ℃ for 8-12 h. After centrifugation, washing, and drying, the product is dispersed in methanol to obtain a ZIF-67 dispersion with a concentration of 0.5-2 mg / mL.

10. The application of the nitrogen and phosphorus co-doped carbon dot @ZIF-67@MIPs composite material according to any one of claims 1-4 in the detection of sulfadiazine, characterized in that, The composite material was mixed with the sample to be tested, and the concentration of sulfadiazine in the actual sample was quantitatively detected using a fluorescence spectrophotometer with an excitation wavelength of 360 nm, based on the standard curve and the change in fluorescence intensity.

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