Ratio-type hollow molecularly imprinted fluorescent encoding microsphere suitable for aqueous phase, preparation method and application of rate-type hollow molecularly imprinted fluorescent encoding microsphere

Abstract

The invention belongs to the technical field of molecular imprinting fluorescence sensing, and discloses a ratio type hollow molecular imprinting fluorescence coding microsphere, a preparation method and application. The method comprises the following steps: (1) preparing a metal organic framework material ZIF-8; (2) preparing a hollow molecularly imprinted polymer HMIPs; (3) preparing single-color hollow molecularly imprinted fluorescent encoding microspheres G-QDs-HMIPs (Gallium Quantitative Density-Hidden Molecular And (4) loading the red quantum dots R-QDs. The coding microspheres prepared by the method have the advantages that the detection in a water phase can be realized; the sensitivity is high, and the repeatability and the stability are good; the detection efficiency is high and the selectivity is good; the detection process is simple; the detection time is short. The coding microspheres are applied to trace detection verification of NOR in milk, chicken, pork and fish meat in food, the result shows that the coding microspheres meet the precision requirement of the international analytical chemistry union for trace analysis, RSD is stabilized within the range of 1.91%-7.56%, and it is proved that a sensing system has excellent repeatability and stability.

Description

Technical Field

[0001] This invention belongs to the field of optical sensing material preparation and rapid food safety detection technology, and specifically relates to a ratiometric hollow molecularly imprinted fluorescently encoded microsphere suitable for aqueous phase, its preparation method and application. Background Technology

[0002] Optical sensors, due to their simplicity, fast response, and low cost, have become one of the most promising analytical technologies in the field of food safety testing. Among the many optical sensing modes, fluorescence sensors have attracted much attention from researchers due to their excellent real-time performance and portability, playing an important role in rapid detection. Ratio-modulated fluorescence sensors, with two or more emission wavelengths, can not only reduce background interference and improve the accuracy of the method through self-calibration, but also improve the naked-eye detection output signal at a very low detection limit. Color changes can be clearly and easily distinguished by the naked eye, enabling reliable, rapid, and visual analysis of the detection method. Combining the selectivity of molecular imprinting technology with the sensitivity and accuracy of ratio-modulated fluorescence sensors, ratio-modulated molecularly imprinted fluorescence sensors have been successfully applied to the rapid detection of small molecule pollutants such as pesticide residues, veterinary drug residues, and biotoxins.

[0003] Hollow-structured materials exhibit significant advantages in specific surface area, loading capacity, and mass transfer efficiency due to their unique cavity structure and adjustable shell. Core-shell molecularly imprinted polymers (MIPs) constructed with hollow structures as the core effectively enhance the adsorption performance of traditional solid MIPs. However, the removal of the core template remains a significant challenge in practical applications. Traditional high-temperature calcination or chemical etching methods easily lead to structural damage and loss of material integrity, hindering their further development. ZIF-8, as a type of material composed of Zn... 2+ Metal-organic frameworks (MOFs) formed by coordination with 2-methylimidazole have become a research hotspot in materials science due to their unique structural properties (including high specific surface area, simple preparation methods, and excellent thermal stability). These materials undergo structural dissociation in acidic media, and this chemical instability allows for effective removal via acidolysis.

[0004] On the other hand, the preparation process of traditional molecularly imprinted fluorescence sensors is mostly carried out in the organic phase. The resulting polymer has strong hydrophobicity, which makes it suitable for detection in the organic phase. However, it requires the use of a large amount of organic solvents, which poses an environmental pollution risk and restricts its practical application potential.

[0005] Norfloxacin (NOR) is a third-generation fluoroquinolone antibiotic. It is commonly used in the treatment of human and animal diseases, particularly infections caused by a variety of susceptible bacteria, because it inhibits the activity of bacterial DNA topoisomerase II (gyrase). Toxicological studies have shown that although NOR has a half-life of only 3-4 hours in the human body, long-term low-dose intake can induce bacterial resistance, lead to intestinal flora imbalance, and potentially cause liver and kidney damage, especially causing irreversible harm to the musculoskeletal development of minors.

[0006] Therefore, it is necessary to establish a detection method with high accuracy and sensitivity for the detection of NOR in food matrices. Summary of the Invention

[0007] To address the aforementioned technical issues and further improve detection efficiency and shorten detection time, this invention focuses on the structure of microspheres, preparing them into hollow molecularly imprinted fluorescently encoded microspheres. This not only enables detection in aqueous phases but also offers advantages such as high sensitivity, good repeatability, excellent stability, simple detection process, high detection efficiency, good selectivity, and short detection time. These advantages have been verified through specific experiments.

[0008] To obtain hollow-structured coded microspheres, this invention uses ZIF-8 as the core, prepares a molecularly imprinted polymer on its surface, and elutes it under acidic conditions. This is because the metal-organic framework ZIF-8 is unstable under acidic conditions. Therefore, a NOR molecularly imprinted layer is prepared on the surface of ZIF-8 as a matrix, and the ZIF-8 matrix is ​​removed under acidic conditions, thus forming a hollow structure. Green and red quantum dots are then loaded using a poly(styrene-maleic anhydride) copolymer (PSMA) and a layer-by-layer assembly method to prepare fluorescently coded microspheres with a hollow structure. This is the most important feature that distinguishes the fluorescently coded microspheres of this invention from other ordinary microspheres. It is precisely because the fluorescently coded microspheres of this invention have the above-mentioned structure that most of their advantages arise from this structure.

[0009] Specifically, this invention first uses PSMA to mediate green quantum dots with an emission wavelength of 525 nm within mesopores, while simultaneously hydrophilically modifying the surface of the hollow imprinted material. Then, through electrostatic self-assembly of polyethyleneimine (PEI), red quantum dots with an emission wavelength of 640 nm are electrostatically modified onto the surface of microspheres via electrostatic interactions, preparing ratiometric hollow molecularly imprinted fluorescently encoded microspheres. These microspheres are then used to construct a fluorescence sensor for the detection of NOR in a food matrix. In practical applications, a dual-channel fluorescence intensity ratio (IL) is established based on the fluorescence intensity response change. 640 / I 525 The quantitative relationship between NOR concentration and NOR concentration was established, enabling dual-signal ratiometric detection of the target analyte.

[0010] The specific technical solution of the present invention is as follows: The ratiometric hollow molecularly imprinted fluorescently encoded microspheres suitable for aqueous phases provided by this invention are prepared by the following method: Using metal-organic framework material ZIF-8 as the core, a molecularly imprinted polymer was prepared on its surface. Under acidic conditions, ZIF-8 and template molecules were removed to obtain a hollow molecularly imprinted layer. Next, the quantum dots with an emission wavelength of 525 nm were encoded into the interior of the molecular imprinted layer using the PSMA-mediated method, while the surface of the imprinted polymer was hydrophilically modified. Then, using a layer-by-layer assembly method, quantum dots with carboxyl groups and an emission wavelength of 640 nm were fixed onto the surface of the molecularly imprinted layer with PEI, and dried to obtain ratiometric hollow molecularly imprinted fluorescently encoded microspheres.

[0011] The quantum dots with an emission wavelength of 525 nm mentioned above are green ZnCdSe / ZnS quantum dots (also referred to as: G-QDs); The quantum dots with an emission wavelength of 640 nm are Cd-MPA red quantum dots (R-QDs) modified with mercaptopropionic acid.

[0012] Specifically, the aforementioned metal-organic framework material ZIF-8 was prepared using the following method: Zinc nitrate and 2-methylimidazole were dissolved in methanol and sonicated to obtain zinc nitrate solution and 2-methylimidazole solution, respectively. The 2-methylimidazole solution was slowly added to the zinc nitrate solution, and the mixture was stirred and reacted at room temperature in the dark for 20-30 h. After centrifugation at 5,000-10,000 rpm for 2-8 min, the precipitate was washed with methanol and dried under vacuum at 35-40 °C to constant weight to obtain the metal-organic framework material ZIF-8. The molar ratio of zinc nitrate to 2-methylimidazole was 1:4.

[0013] The above-described method for preparing ratiometric hollow molecularly imprinted fluorescently encoded microspheres suitable for aqueous phases includes the following steps: (1) Preparation of metal-organic framework material ZIF-8 (2) Preparation of hollow molecularly imprinted polymers (HMIPs) Take the metal-organic framework material ZIF-8 obtained in (1), add it to the porogen, disperse it by ultrasonication, add the template molecule, dissolve it by ultrasonication, add the functional monomer, crosslinking agent and initiator, disperse it by ultrasonication, pass nitrogen gas to remove oxygen, heat it under nitrogen protection, polymerize it, wash the obtained polymer with acetonitrile, filter it, dry it to constant weight (ZIF-8@MIPs), then elute it with methanol / acetic acid solution to remove the template molecule and ZIF-8, dry it, and obtain the molecularly imprinted polymer HMIPs with hollow structure; The mass-to-volume ratio of ZIF-8 to the porogen was 100.0 mg:(15-25) mL; The ratio of template molecule to ZIF-8 was 0.1 mmol: 100.0 mg; The molar ratio of template molecule: functional monomer: crosslinking agent is 1:4:10; the ratio of template molecule to initiator is 0.1 mmol: 20.0 mg. The crosslinking agent is composed of ethylene glycol dimethacrylate and divinylbenzene, with a molar ratio of ethylene glycol dimethacrylate to divinylbenzene of 1:1; (3) Preparation of monochromatic hollow molecularly imprinted fluorescently encoded microspheres G-QDs-HMIPs Green ZnCdSe / ZnS quantum dots G-QDs were dispersed in the organic solvent n-hexane; The coding matrix HMIPs and PSMA were added sequentially, and the mixture was ultrasonically treated to form a homogeneous dispersion system. Then, NaOH solution was immediately added, and ultrasonic emulsification was used to promote the microspheres to change from the oil phase to the aqueous phase. After centrifugation, washing, and drying, monochromatic molecularly imprinted fluorescent coding microspheres G-QDs-HMIPs were obtained. (4) Loading red quantum dots R-QDs modified with mercaptopropionic acid Dissolve PEI in MEST buffer (10 mmol / L) -1 In a solution of pH 5.0, add the monochromatic molecularly imprinted fluorescently encoded microspheres G-QDs-HMIPs prepared in (3), disperse by ultrasonication, react, centrifuge, and collect the PEI-modified G-QDs-HMIPs microspheres; G-QDs-HMIPs microspheres and mercaptopropionic acid-modified red quantum dots R-QDs were dispersed in MEST (100 mmol / L) respectively. -1 In a buffer solution (pH 6.5), the dispersion of the former was added to the dispersion of the latter, and the reaction was carried out. The quantum dots were oriented and assembled by electrostatic interaction. After the reaction was completed, the particles were centrifuged, washed, and dried to obtain ratiometric hollow molecularly imprinted fluorescently encoded microspheres G-QDs-HMIPs-R-QDs with dual emission signals.

[0014] Preferably, in (2), the porogen is acetonitrile; the template molecule is norfloxacin; The functional monomer is methacrylic acid; the initiator is azobisisobutyronitrile; the volume ratio of methanol to acetic acid in the methanol / acetic acid solution is 8:(1-5); Preferably, in (3), the organic solvent is n-hexane; 10.0 mg of HMIPs were dispersed in 1 mL of n-hexane, followed by the addition of 50 μL of green quantum dots G-QDs and 5.0 mg of PSMA. The mixture was sonicated for 10 min to form a homogeneous dispersion. Then, 500 μL of 1 mol L⁻¹ was immediately added. -1 The microspheres were ultrasonically emulsified in NaOH solution to facilitate the transfer of the microspheres from the oil phase to the aqueous phase. Finally, the microspheres were centrifuged at 10,000 rpm for 5 min, washed three times with deionized water, and then freeze-dried under vacuum at -50℃ for 4 h to obtain G-QDs-HMIPs microspheres. Preferably, in (4), the MEST buffer is an aqueous solution of 2-(N-morpholino)ethanesulfonic acid (MES) containing 0.05% Tween-20 (w / v); The mass-to-volume ratio of PEI to MEST buffer was 150.0 mg : 2.6 mL; the molecular weight of PEI was 10 kDa; the concentration of MEST buffer was 10 mmol / L. -1 pH 5.0; The mass ratio of microspheres G-QDs-HMIPs to PEI was 10.0 mg: 150.0 mg. The reaction time is 30 min.

[0015] In (4), to enhance the stability of interfacial bonding, 1 mL of MEST (100 mmol / L) containing 10.0 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 10.0 mg of N-hydroxysuccinimide (NHS) was added to the reaction system during the reaction to achieve directional assembly. -1 React at room temperature for 3 hours; After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 5 min and washed five times with deionized water to remove unbound components. The final product was freeze-dried at -50℃ for 4 h to obtain G-QDs-HMIPs-R-QDs encoded microspheres with dual emission signals.

[0016] The application of the aforementioned ratiometric hollow molecularly imprinted fluorescently encoded microspheres in the detection of trace amounts of norfloxacin in food is also within the scope of protection of this invention. The above-mentioned foods can be any of chicken, pork, fish, or milk, or other foods containing trace amounts of norfloxacin, and are not limited to the foods listed above.

[0017] The ratiometric hollow molecularly imprinted fluorescently encoded microspheres prepared in this invention not only reduce material density, effectively improve adsorption performance and fluorescence intensity, and shorten detection time, but also utilize layer-by-layer assembly technology to load two types of quantum dots, thus avoiding the influence of molecular imprint layer coating on the fluorescence intensity of the internal quantum dots. More importantly, the fabrication process of traditional molecularly imprinted fluorescent sensors is mostly carried out in organic phases, resulting in polymers with strong hydrophobicity. This makes them suitable for detection in organic phases, requiring large amounts of organic solvents and posing a significant environmental pollution risk. This limits the application of this method, and in the long run, it will likely be abandoned or replaced. Therefore, in this invention, microspheres obtained by hydrophilically modifying the polymer surface can be used for detection in aqueous systems, avoiding the use of organic reagents during the detection process. This not only greatly reduces the risk of environmental pollution but also reduces the high detection costs associated with using organic solvents.

[0018] In the preparation of ratiometric hollow molecularly imprinted fluorescently encoded microspheres, this invention utilizes the instability of the metal-organic framework ZIF-8 under acidic conditions. A NOR molecularly imprinted layer is prepared on the surface of ZIF-8 as a substrate, and the ZIF-8 substrate is removed under acidic conditions to form a hollow structure. Two colors of quantum dots are then loaded using a layer-by-layer assembly method to prepare fluorescently encoded microspheres. The encoded microspheres obtained by the above method have the following characteristics: (1) It can be detected in aqueous phase. Compared with traditional molecularly imprinted fluorescent sensors, the ratiometric hollow molecularly imprinted fluorescently encoded microspheres prepared in this invention utilize layer-by-layer assembly technology to modify the surface of the microspheres with hydrophilicity while loading quantum dots, thus enabling detection in the aqueous phase, avoiding the use of large amounts of organic solvents, and greatly reducing environmental pollution. (2) High sensitivity, good repeatability and stability The recovery rates of spiked samples for testing milk, chicken, pork, and fish ranged from 91.04% to 104.63%, with relative standard deviations (RSDs) all below 7.56% (n=3), meeting the precision requirements (RSD < 10%) for trace analysis set by the International Union of Analytical Chemistry (IUPAC). The RSDs for intra-day precision (3 consecutive measurements within 1 day) and inter-day precision (3 consecutive measurements over 3 days) remained stable within the range of 1.91% to 7.56%, confirming that the sensing system has excellent repeatability and stability. (3) The detection process is simple and the detection efficiency is high. After the sample is prepared, simply add the G-QDs-HMIPs-R-QDs fluorescently encoded microspheres of this invention to the test solution, and disperse by ultrasonication to obtain a final concentration of 1 mg·mL⁻¹ for the fluorescent sensor. -1 After shaking in the dark at room temperature for 8 minutes to allow for a complete reaction, the fluorescence intensity of each sample solution is measured using a fluorescence spectrophotometer, thus enabling trace detection of NOR in food. This invention loads two quantum dots with different emission wavelengths into a hollow molecularly imprinted polymer with low density and good adsorption performance to prepare ratiometric hollow molecularly imprinted fluorescently encoded microspheres. By using layer-by-layer assembly technology to load quantum dots, the precise functional design of the molecular imprinted layer is achieved. The hollow structure of the encoded microspheres greatly improves their adsorption performance, thereby improving detection efficiency and fast adsorption rate. (4) Good selectivity Because the red quantum dots (R-QDs) modified with mercaptopropionic acid loaded on the outer layer of the molecular imprint in this invention have a certain selectivity for the target molecules, and the specific adsorption sites of the molecular imprint endow them with a certain targeting ability, they can achieve dual recognition of the target. This makes the ratiometric hollow molecular imprinted fluorescently encoded microspheres have excellent selectivity, and the microspheres also have strong resistance to external interference. For specific verification, see Example 5. (5) Short detection time Because the coded microspheres in this invention have a hollow structure, they are characterized by a fast mass transfer rate. Therefore, in the actual detection process of NOR, the detection time can be shortened to 8 minutes, which is much shorter than the traditional detection method.

[0019] In summary, the coded microspheres of this invention enable rapid identification and quantitative analysis of NOR in milk, chicken, pork, and fish. Attached Figure Description

[0020] Figure 1 Fourier transform infrared spectra of HMIPs, G-QDs-HMIPs, and G-QDs-HMIPs-R-QDs microspheres; Figure 2 Transmission electron micrographs of (a) ZIF-8@MIPs and (b) HMIPs; Figure 3 XRD patterns of ZIF-8, ZIF-8@MIPs and HMIPs; Figure 4 Adsorption-desorption isotherms for HMIPs and S-MIPs microspheres; Figure 5 The changes in fluorescence intensity and fluorescence response of microspheres with different amounts of G-QDs added before and after the addition of NOR; Figure 6 The changes in fluorescence intensity and fluorescence response of microspheres with different amounts of R-QDs added before and after the addition of NOR; Figure 7 Zeta potential diagrams for G-QDs-HMIPs, PEI-modified G-QDs-HMIPs, and G-QDs-HMIPs-R-QDs microspheres; Figure 8 Natural light photographs of HMIPs (left) and G-QDs-HMIPs-R-QDs (right) in aqueous solution; Figure 9 The response changes of NOR to the G-QDs-HMIPs-R-QDs fluorescence sensor under different response times; Figure 10 (a) and (c) fluorescence responses of R-QDs and G-QDs-HMIPs-R-QDs to NOR and its structural analogs, tetracyclines, amino acids and (b) and (d) common ions, respectively; Figure 11 The normalized fluorescence intensity data are shown in the figure. (a) shows the fluorescence intensity changes of G-QDs-HMIPs-R-QDs microspheres, G-QDs, and R-QDs after irradiation for different durations at different UV wavelengths; (b) shows the fluorescence intensity changes of G-QDs-HMIPs-R-QDs microspheres at 640 nm UV wavelength under different pH conditions. Figure 12 This is a schematic diagram of the preparation process of ratiometric hollow molecularly imprinted fluorescently encoded microspheres. Detailed Implementation

[0021] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0022] In this invention, both the green quantum dots G-QDs and the red quantum dots R-QDs modified with mercaptopropionic acid are commercially available products, and both are manufactured by Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd.

[0023] Example 1-1 A method for preparing ratiometric hollow molecularly imprinted fluorescently encoded microspheres suitable for aqueous phases includes the following steps: (1) Dissolve 0.7344 g of zinc nitrate and 0.8106 g of 2-methylimidazole in 50 mL of methanol respectively, and sonicate to dissolve them to obtain zinc nitrate solution and 2-methylimidazole solution. Slowly add 2-methylimidazole solution to zinc nitrate solution and stir at room temperature in the dark for 24 h. Centrifuge the resulting suspension at 8,000 rpm for 5 min to obtain precipitate. Wash the precipitate 3 times with methanol and dry it in a vacuum drying oven at 40℃ for 8 h to obtain metal-organic framework material ZIF-8. (2) Using the metal-organic framework material ZIF-8 from (1) as the core, 100.0 mg of ZIF-8 was dispersed in 20 mL of acetonitrile solution and sonicated for 10 min to form a uniform suspension. Norfloxacin (NOR, 0.1 mmol) was added as the template molecule and dissolved by sonication. Functional monomers (methacrylic acid, 0.4 mmol), crosslinking agents (ethylene glycol dimethacrylate, 1 mmol; divinylbenzene, 1 mmol) and initiator (azobisisobutyronitrile, 20.0 mg) were added. After sonication for 5 min, nitrogen gas was introduced for 15 min to remove oxygen. The mixture was then sealed under nitrogen protection and polymerized in a water bath at 60 °C for 24 hours. h, the obtained polymer was washed with acetonitrile to remove unreacted reagents, filtered, and dried at 60°C to constant weight (ZIF-8@MIPs). Then, a methanol / acetic acid mixed solvent (8:2, v / v) was added for Soxhlet extraction and elution until the template was removed. At the same time, the core ZIF-8 was dissolved in its acidic environment. After drying to constant weight, hollow molecularly imprinted polymers (HMIPs) were obtained. (3) Disperse 50 μL of green quantum dots G-QDs (commercially available product) in 1 mL of n-hexane, then add HMIPs (10.0 mg) and PSMA (5.0 mg) prepared in (2) sequentially, sonicate for 10 min to form a homogeneous dispersion system, and then immediately add 500 μL of NaOH solution (1 mol L). -1 The microspheres were ultrasonically emulsified for 5-10 minutes to promote the transfer of the microspheres from the oil phase to the aqueous phase. Finally, they were centrifuged at 10,000 rpm for 5 minutes, washed three times with deionized water, and then freeze-dried in vacuum at -50℃ for 4 hours to obtain G-QDs-HMIPs microspheres. (4) The R-QDs were loaded using a layer-by-layer assembly method. The specific experimental steps are as follows: First, the obtained G-QDs-HMIPs microspheres were amino-functionalized using PEI. The mixture was then placed in 2.6 mL of MEST buffer (10 mmol / L). -1Dissolve 150.0 mg of PEI (Mw = 10 kDa) in water (pH 5.0), add 10.0 mg of G-QDs-HMIPs microspheres, disperse by sonication, and rotate in a mixer for 30 min to complete the surface functionalization of the microspheres. After the reaction is complete, collect the PEI-modified G-QDs-HMIPs microspheres by centrifugation. (5) The G-QDs-HMIPs microspheres obtained above and 70 μL of MPA-modified red quantum dots R-QDs were dispersed in 300 μL of high-concentration MEST buffer (100 mmol / L). -1 After sonicating for 10 min to form a stable dispersion in a solution of G-QDs-HMIPs (pH 6.5), the G-QDs-HMIPs dispersion was slowly added to the R-QDs solution under ultrasonic assistance. The mixture was then rotated in a mixer for 30 min to achieve the directional assembly of quantum dots via electrostatic interactions. To enhance interfacial bonding stability, 1 mL of MEST buffer (100 mmol / L) containing EDC (10.0 mg) and NHS (10.0 mg) was added to the reaction system. -1 The mixture was reacted at room temperature for 3 h. After the reaction, the mixture was centrifuged at 10,000 rpm for 5 min and washed five times with deionized water to remove unbound components. The final product was freeze-dried at -50℃ for 4 h to obtain G-QDs-HMIPs-R-QDs encoded microspheres with dual emission signals.

[0024] To better understand the performance of the encoded microspheres provided by this invention, the obtained ZIF-8, HMIPs, G-QDs-HMIPs, and G-QDs-HMIPs-R-QDs encoded microspheres were characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), X-ray diffraction (XRD), and nitrogen adsorption-desorption (BET), respectively. The results are shown in […]. Figures 1-4 .

[0025] from Figure 1 It can be seen that 1449 cm in HMIPs -1 The characteristic peak appearing at 1721 cm⁻¹ is attributed to the C-S stretching vibration peak of the benzene ring in divinylbenzene. -1 The absorption peak at 1772 cm⁻¹ corresponds to the C=O stretching vibration peak of the carboxylic acid group of methacrylic acid, confirming the successful introduction of the functional monomer and crosslinking agent. In G-QDs-HMIPs microspheres, the absorption peak at 1772 cm⁻¹ corresponds to the C=O stretching vibration peak of the carboxylic acid group of methacrylic acid, confirming the successful introduction of the functional monomer and crosslinking agent. -1 With 1847 cm -1The observation of symmetric and asymmetric stretching vibration characteristic peaks of the anhydride groups (C=O) in PSMA indicates that PSMA was successfully incorporated into the material, and that the anhydride groups underwent hydrolysis under the action of NaOH. Further spectral analysis of the G-QDs-HMIPs-R-QDs microspheres revealed peaks at 2840 cm⁻¹. -1 A peak of methylene (CH2) symmetric stretching vibration appears at 1559 cm⁻¹. -1 The appearance of bending vibration peaks of primary amine (-NH-) groups confirms that PEI was successfully assembled onto the material surface.

[0026] Depend on Figure 2 (a) It can be seen that, without eluting the template molecules, the ZIF-8 surface successfully coated with a molecularly imprinted polymer layer, forming ZIF-8@MIPs with a clearly visible core-shell structure. After acid elution, the ZIF-8 material dissociates under acidic conditions, forming a hollow structure inside, while maintaining an intact imprinted layer (see...). Figure 2 (b) proves the successful preparation of the hollow structure.

[0027] Depend on Figure 3 As shown, the characteristic diffraction peaks of the original ZIF-8 are in excellent agreement with the ZIF-8 simulation data obtained from the Cambridge Structural Data Center (CCDC 602542). Furthermore, the characteristic diffraction peaks of ZIF-8 are still clearly observed in the core-shell structured ZIF-8@MIPs microspheres, indicating that ZIF-8 has been successfully incorporated into the material. After acid elution, the characteristic peaks of ZIF-8 completely disappeared in the XRD pattern of HMIPs, indicating the complete removal of ZIF-8 material under acidic conditions. This result not only matches the TEM results but also directly proves the formation of the hollow structure.

[0028] Depend on Figure 4 As can be seen from the Brunauer-Emmett-Teller (BET) theory, the specific surface area of ​​the HMIPs prepared in this invention is 12.6 m². 2 g -1 .

[0029] Examples 1-2 The difference from Example 1-1 is that in step (3), the amount of G-QDs added is changed from 50 μL to 10, 20, 30, 70, 100, and 150 μL, while the other steps are exactly the same as in Example 1-1; Figure 5 It can be seen that when the amount of G-QDs added is 10 μL-50 μL, the fluorescence quenching efficiency gradually increases with the increase of the amount of G-QDs added, reaching a maximum value when the amount added is 50 μL, and then gradually weakening with the increase of the amount of G-QDs added. This indicates that, in comparison, 50 μL in Example 1-1 is a more optimized amount of G-QDs added.

[0030] Examples 1-3 The difference from Example 1-1 is that the amount of MPA-modified red quantum dots added in step (5) is changed from 70 μL to 20, 50, and 100 μL, while the other steps are exactly the same as in Example 1-1; Figure 6 It can be seen that when the amount of red quantum dots added is 20, 50, and 100 μL, the change in fluorescence response is lower than that when the amount added is 70 μL (the change in fluorescence response reaches the maximum value of 1.52). This indicates that, compared to the G-QDs-HMIPs-R-QDs microspheres prepared by adding 70 μL of red quantum dots in Examples 1-1, the fluorescence response is better. The red quantum dots modified with mercaptopropionic acid are also a relatively key parameter of this invention. If the amount added is inappropriate, the fluorescence response effect will be unsatisfactory.

[0031] Example 2 Based on Example 1-1, the zeta potential of G-QDs-HMIPs-R-QDs microspheres was analyzed.

[0032] like Figure 7 As shown, during the mediated modification of HMIPs, the Zeta potential shifted negatively to -54.71 mV. This electronegativity shift is attributed to the hydrolysis of the anhydride groups in the PSMA molecule under alkaline conditions, exposing a large number of negatively charged carboxylic acid groups (-COOH), resulting in a strongly electronegative surface. After PEI modification, the surface potential reversed, jumping from the initial -54.71 mV to +52.29 mV. This result directly confirms that the abundant amino groups (-NH2) in the PEI molecule were successfully grafted onto the material surface, forming a positively charged functional layer. Finally, when the negatively charged R-QDs bonded to the PEI-modified layer through electrostatic interactions, the Zeta potential in the composite system decreased to +47.67 mV. This regular change confirms the electrostatic self-assembly mechanism between the R-QDs and the PEI-modified layer, providing a reliable surface chemical basis for the construction of dual quantum dot-encoded microspheres.

[0033] Example 3 Based on Example 1-1, the surface modification strategy of G-QDs-HMIPs-R-QDs microspheres was analyzed.

[0034] HMIPs and G-QDs-HMIPs-R-QDs microspheres were dispersed in aqueous solutions, and the dispersibility of the materials in water was observed under natural light conditions. The results are as follows: Figure 8 As shown.

[0035] Dispersion experiments in aqueous solution show that ( Figure 8Unmodified HMIPs exhibit strong hydrophobicity due to the hydrophobic effect of the benzene ring in the crosslinking agent divinylbenzene, and thus aggregate in aqueous solution. In contrast, G-QDs-HMIPs-R-QDs microspheres, after carboxyl functionalization, demonstrate excellent aqueous dispersibility. This proves that the surface modification strategy effectively improves the hydrophilicity of the material, and the resulting G-QDs-HMIPs-R-QDs microspheres are suitable for use in aqueous systems.

[0036] Example 4 Based on Example 1-1, the response time of G-QDs-HMIPs-R-QDs microspheres to the target NOR was optimized.

[0037] G-QDs-HMIPs-R-QDs encoded microspheres were dispersed in water (concentration of 1.0 mg / mL). -1 Then, NOR standard solution was added to make the concentration of NOR in the encoded microsphere solution 100 μg / L. -1 The microspheres were placed in an oscillator and oscillated for 2, 4, 6, 8, 10, 15, and 20 minutes, respectively. Fluorescence intensity was measured using a fluorescence spectrophotometer to evaluate the effect of different response times on the fluorescence response of the encoded microspheres. The results are as follows: Figure 9 As shown.

[0038] like Figure 9 As shown, the fluorescence response of G-QDs-HMIPs-R-QDs microspheres increased continuously after the addition of NOR, reaching equilibrium at 8 min. Even with further extension of the response time, no significant change in the microsphere response was observed. To shorten the analysis time, 8 min was ultimately chosen as the detection time. More importantly, compared to the response time of the solid core-shell imprinted fluorescence sensor used in previous studies (15 min), this is approximately half the time (Patent No.: 2024111804950). This is mainly due to the low density of the hollow structure material, which allows for a greater loading of target components per unit mass, thus improving adsorption efficiency and demonstrating significant advantages in accelerating mass transfer rates and enhancing adsorption capacity.

[0039] Example 5 Based on Examples 1-1, the selectivity and anti-interference ability of R-QDs and G-QDs-HMIPs-R-QDs microspheres for the target NOR were evaluated.

[0040] R-QDs and G-QDs-HMIPs-R-QDs encoded microspheres were dispersed in water (concentration 1.0 mg / mL). -1Then, standard solutions of NOR, NOR structural analogues (ofloxacin OFX, enrofloxacin ENX, ciprofloxacin CIP), tetracycline (TC), amino acids (phenylalanine Phe, leucine Leu, glycine Gly, cysteine ​​Cys, and methionine Met) and ions (Al) were added respectively. 3+ Ca 2+ Fe 2+ Fe 3+ K + Na + Zn 2+ Cl - and NO3 - Each concentration was 100 μg / L. -1 Compare the responses to different fluorescence intensities.

[0041] like Figure 10 As shown, the fluorescence quenching efficiency of NOR on R-QDs reached 1.45, higher than that of its structural analogs OFX, ENX, CIP, and the common antibiotic TC. Other coexisting interfering compounds showed only fluorescence response changes of 0.98-1.05, demonstrating that R-QDs themselves possess a certain selectivity for NOR. In contrast, G-QDs-HMIPs-R-QDs microspheres exhibited stronger selectivity for NOR than their structural analogs. The fluorescence response changes of coexisting compounds such as antibiotics, amino acids, and ions ranged from 0.966 to 1.028. These results indicate that the prepared ratiometric fluorescence sensor possesses excellent selectivity and anti-interference capabilities, avoiding interference from complex food matrices on the detection system.

[0042] Example 6 Based on Example 1-1, the photostability and pH stability of G-QDs-HMIPs-R-QDs microspheres were evaluated.

[0043] 1.0 mg mL -1 G-QDs-HMIPs-R-QDs microspheres and G-QDs and R-QDs quantum dots were continuously irradiated with 365 nm ultraviolet light for 24 h, and the fluorescence intensity of different systems at different time intervals was monitored. Simultaneously, 1.0 mg of G-QDs-HMIPs-R-QDs microspheres were dispersed in 1 mL solutions of different pH values, and the fluorescence intensity change of the G-QDs-HMIPs-R-QDs microspheres at 640 nm was measured at different pH values.

[0044] like Figure 11As shown, after 24 h of UV irradiation, the fluorescence intensity of both G-QDs and R-QDs decreased compared to the original values ​​(to 31.06% and 28.84% of the initial values, respectively), while in the G-QDs-HMIPs-R-QDs microspheres, the fluorescence intensity remained above 93.51% and 87.25% of the initial values, respectively. Simultaneously, the fluorescence intensity of the G-QDs-HMIPs-R-QDs microspheres remained at 95.41% of the maximum value within the pH range of 3.0-14.0. These results indicate that the prepared G-QDs-HMIPs-R-QDs microspheres possess photostability and exhibit good fluorescence stability in weakly acidic, neutral, and strongly alkaline environments.

[0045] Example 7 A fluorescence sensor was constructed using ratiometric hollow molecularly imprinted fluorescently encoded microspheres as the matrix through spiked recovery experiments. The recovery rates of NOR in four food samples (chicken, pork, fish, and milk) were determined to verify the sensor's accuracy in real samples. Three spiked concentration gradients (5, 10, and 50 μg kg) were used. -1 / μg L -1 The accuracy of the recovery experiment evaluation system.

[0046] A NOR fluorescence sensor was constructed using ratiometric hollow molecularly imprinted fluorescently encoded microspheres for the detection of NOR in milk, chicken, pork, and fish. The feasibility of the fluorescence sensor in the detection of actual samples was evaluated through a spiked recovery experiment.

[0047] For chicken, pork, and fish samples, accurately weigh 5.0 g of sample into 50 mL PTFE centrifuge tubes, and add NOR to achieve final sample concentrations of 5, 10, and 50 μg / L, respectively. -1 After standing for equilibration for 12 h, 10.0 mL of aqueous solution was added as the extraction solvent; the mixture was immediately vortexed for 2 min, then vortexed again for 2 min in a 30℃ water bath for 10 min, and centrifuged at 7,000 rpm for 5 min to collect the supernatant. The extraction was repeated twice and the supernatants were combined.

[0048] Milk samples were processed using the same procedure, with the addition of 10.0 μL of acetic acid solution to remove protein interference. The processed sample was used as the test solution. G-QDs-HMIPs-R-QDs fluorescently encoded microspheres were added to the test solution and ultrasonically dispersed to obtain the fluorescence sensor. The final concentration of the sensor was 1 mg·mL⁻¹. -1 The samples were shaken in the dark at room temperature for 8 minutes to allow for a complete reaction. The fluorescence intensity of each sample solution was then measured using a fluorescence spectrophotometer. The results are shown in Table 1.

[0049] Table 1. Recovery rate and RSD of NOR detection in different samples

[0050] ND a Not detected The constructed fluorescence sensor was successfully applied to the trace detection of NOR in milk, chicken, pork, and fish. The recoveries of spiked samples ranged from 91.04% to 104.63%, and the relative standard deviations (RSDs) were all below 7.56% (n=3), meeting the precision requirements (RSD < 10%) for trace analysis set by the International Union of Analytical Chemistry (IUPAC). The RSDs for intra-day precision (3 consecutive measurements within one day) and inter-day precision (3 consecutive measurements over 3 days) remained stable within the range of 1.91% to 7.56%, confirming that the sensing system has excellent repeatability and stability.

[0051] In summary, this invention successfully prepared hollow structure imprinted coding microspheres by removing the ZIF-8 core under acidic conditions. Furthermore, through PSMA-mediated directional assembly of G-QDs and layer-by-layer modification of R-QDs surface fixation technology, the precise functional design of molecular imprinted layers was achieved.

[0052] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

[0053] Comparative Example 1 Based on Example 1-1, without the addition of ZIF-8, solid molecularly imprinted polymers (S-MIPs) without hollow structures were directly polymerized to evaluate the effect of hollow structures on adsorption performance.

[0054] The S-MIPs prepared in this comparative example were characterized by N2 adsorption-desorption desorption. The characterization results are shown in the figure. Figure 4 .

[0055] like Figure 4 As shown, based on the Brunauer-Emmett-Teller (BET) theory, the specific surface area of ​​S-MIPs is 8.6 m². 2 g -1 .

[0056] Compared with the specific surface area of ​​HMIPs in Example 1-1 (12.6 m²), 2 g -1 Compared to HMIPs, S-MIPs have a significantly lower specific surface area. The higher specific surface area also indicates that HMIPs have better adsorption capacity, can adsorb more template molecules, and load more QDs in polymer microspheres.

[0057] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A ratiometric hollow molecularly imprinted fluorescently encoded microsphere suitable for aqueous phases, characterized in that, The microspheres were prepared by the following method: Using metal-organic framework material ZIF-8 as the core, a molecularly imprinted polymer was prepared on its surface. Under acidic conditions, ZIF-8 and template molecules were eluted to remove them, resulting in a hollow molecularly imprinted layer. Next, the quantum dots with an emission wavelength of 525 nm were encoded into the interior of the molecular imprinted layer using the poly(styrene-maleic anhydride) copolymer PSMA-mediated method, while the surface of the imprinted polymer was hydrophilically modified. Then, using a layer-by-layer assembly method, quantum dots with carboxyl groups and an emission wavelength of 640 nm were fixed onto the surface of the molecularly imprinted layer with polyethyleneimine (PEI), and dried to obtain ratiometric hollow molecularly imprinted fluorescently encoded microspheres.

2. The ratiometric hollow molecularly imprinted fluorescently encoded microspheres suitable for aqueous phases as described in claim 1, characterized in that, The quantum dots with an emission wavelength of 525 nm are green ZnCdSe / ZnS quantum dots G-QDs; The quantum dots with an emission wavelength of 640 nm are red quantum dots (R-QDs) modified with mercaptopropionic acid.

3. The ratiometric hollow molecularly imprinted fluorescently encoded microspheres suitable for aqueous phases as described in claim 1, characterized in that, The metal-organic framework material ZIF-8 was prepared by the following method: Zinc nitrate and 2-methylimidazole were dissolved in methanol and sonicated to obtain zinc nitrate solution and 2-methylimidazole solution, respectively. The 2-methylimidazole solution was slowly added to the zinc nitrate solution, and the mixture was stirred and reacted at room temperature in the dark for 20-30 h. After centrifugation at 5,000-10,000 rpm for 2-8 min, the precipitate was washed with methanol and dried under vacuum at 35-40℃ to constant weight to obtain the metal-organic framework material ZIF-8. The molar ratio of zinc nitrate to 2-methylimidazole was 1:

4.

4. The method for preparing ratiometric hollow molecularly imprinted fluorescently encoded microspheres suitable for aqueous phase as described in claim 1, comprising the following steps: (1) Preparation of metal-organic framework material ZIF-8 (2) Preparation of hollow molecularly imprinted polymers (HMIPs) Take the metal-organic framework material ZIF-8 obtained in (1), add it to the porogen, disperse it by ultrasonication, add the template molecule, dissolve it by ultrasonication, add the functional monomer, crosslinking agent and initiator, disperse it by ultrasonication, pass nitrogen gas to remove oxygen, heat it under nitrogen protection, polymerize it, wash the obtained polymer with acetonitrile, filter it, dry it to constant weight (ZIF-8@MIPs), then elute it with methanol / acetic acid solution to remove the template molecule and ZIF-8, dry it, and obtain the molecularly imprinted polymer HMIPs with hollow structure; in, The mass-to-volume ratio of ZIF-8 to the porogen is 100.0 mg: (15-25) mL; The ratio of template molecule to ZIF-8 is: 0.1 mmol: 100.0 mg; The molar ratio of template molecule: functional monomer: crosslinking agent is 1:4:10; the ratio of template molecule to initiator is 0.1 mmol: 20.0 mg. The crosslinking agent is composed of ethylene glycol dimethacrylate and divinylbenzene, with a molar ratio of ethylene glycol dimethacrylate to divinylbenzene of 1:1; (3) Preparation of monochromatic hollow molecularly imprinted fluorescently encoded microspheres G-QDs-HMIPs Green ZnCdSe / ZnS quantum dots G-QDs were dispersed in the organic solvent n-hexane; The coding matrix HMIPs and PSMA were added sequentially, and the mixture was ultrasonically treated to form a homogeneous dispersion system. Then, sodium hydroxide (NaOH) solution was immediately added, and ultrasonic emulsification was used to promote the microspheres to change from the oil phase to the aqueous phase. After centrifugation, washing, and drying, monochromatic molecularly imprinted fluorescently encoded microspheres G-QDs-HMIPs were obtained. (4) Loading red quantum dots R-QDs modified with mercaptopropionic acid Dissolve PEI in MEST buffer (10 mmol / L) -1 In a solution of pH 5.0, add the monochromatic molecularly imprinted fluorescently encoded microspheres G-QDs-HMIPs prepared in (3), disperse by ultrasonication, react, centrifuge, and collect the PEI-modified G-QDs-HMIPs microspheres; G-QDs-HMIPs microspheres and mercaptopropionic acid-modified red quantum dots R-QDs were dispersed in MEST buffer (100 mmol / L). -1 In a pH 6.5 environment, the dispersion of the former was added to the dispersion of the latter, and the reaction was carried out. The quantum dots were oriented and assembled by electrostatic interaction. After the reaction was completed, the mixture was centrifuged, washed, and dried to obtain ratiometric hollow molecularly imprinted fluorescently encoded microspheres G-QDs-HMIPs-R-QDs with dual emission signals.

5. The method for preparing ratiometric hollow molecularly imprinted fluorescently encoded microspheres suitable for aqueous phases as described in claim 4, characterized in that, In (2), the pore-forming agent is acetonitrile; The template molecule is norfloxacin; The functional monomer is methacrylic acid; The initiator is azobisisobutyronitrile; The volume ratio of methanol to acetic acid in the methanol / acetic acid solution is 8:(1-5).

6. The method for preparing ratiometric hollow molecularly imprinted fluorescently encoded microspheres suitable for aqueous phases as described in claim 4, characterized in that, In (3), the organic solvent is n-hexane; 10.0 mg of HMIPs were dispersed in 1 mL of n-hexane, followed by the addition of 50 μL of green quantum dots G-QDs and 5.0 mg of PSMA. The mixture was sonicated for 10 min to form a homogeneous dispersion. Then, 500 μL of 1 mol L⁻¹ HMIPs was immediately added. -1 The microspheres were ultrasonically emulsified in a sodium hydroxide (NaOH) solution to facilitate the transfer of the microspheres from the oil phase to the aqueous phase. Finally, the microspheres were centrifuged at 10,000 rpm for 5 min, washed three times with deionized water, and then freeze-dried under vacuum at -50℃ for 4 h to obtain G-QDs-HMIPs microspheres.

7. The method for preparing ratiometric hollow molecularly imprinted fluorescently encoded microspheres suitable for aqueous phases as described in claim 4, characterized in that, (4) The MEST buffer is an aqueous solution of 2-(N-morpholino)ethanesulfonic acid (MES) containing 0.05% Tween-20 (w / v); The mass-to-volume ratio of PEI to MEST buffer was 150.0 mg : 2.6 mL; the molecular weight of PEI was 10 kDa; the concentration of MEST buffer was 10 mmol / L. -1 pH 5.0; The mass ratio of microspheres G-QDs-HMIPs to PEI was 10.0 mg: 150.0 mg. The reaction time is 30 min.

8. The method for preparing ratiometric hollow molecularly imprinted fluorescently encoded microspheres suitable for aqueous phases as described in claim 4, characterized in that, In (4), during the reaction to achieve directed assembly, 1 mL of MEST activation solution (100 mmol / L) containing 10.0 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 10.0 mg of N-hydroxysuccinimide (NHS) was added to the reaction system. -1 React at room temperature for 3 hours; After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 5 min and washed five times with deionized water to remove unbound components. The final product was freeze-dried at -50℃ for 4 h to obtain G-QDs-HMIPs-R-QDs encoded microspheres with dual emission signals.

9. The application of the ratiometric hollow molecularly imprinted fluorescently encoded microspheres according to any one of claims 1-3 in the detection of trace amounts of norfloxacin in food.

10. The application as described in claim 9, characterized in that, The food product is any one of chicken, pork, fish, or milk.

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