Ratio type core-shell molecularly imprinted quantum dot fluorescence encoding microsphere, preparation method and application

Abstract

The invention belongs to the technical field of molecular imprinting fluorescence sensing, and discloses a ratio type core-shell molecular imprinting quantum dot fluorescence encoding microsphere, a preparation method and application. The preparation method comprises the following steps: firstly, mixing a metal organic framework material ZIF-8 with green quantum dots for primary coding to obtain a carrier ZIF-8 green quantum dot fluorescence coding material, synthesizing a norfloxacin molecular imprinting shell layer on the surface of the carrier, and then mixing with red quantum dots for secondary coding to obtain the ratio type core-shell molecular imprinting quantum dot fluorescence coding microspheres. And by combining with an organic solvent, the constructed related sensor is used for rapid detection of norfloxacin. The method disclosed by the invention has the advantages that the core-shell structure can avoid interference of two quantum dots on fluorescence properties caused by contact, and the existence of the ZIF-8 core material can increase the specific surface area of the encoding microspheres, accelerate the mass transfer rate, greatly shorten the detection time and improve the detection accuracy. And a new solution is provided for rapid quantitative detection of norfloxacin in a food matrix.

Description

Technical Field

[0001] The invention belongs to the technical field of optical sensing material preparation and food safety detection, and particularly relates to a ratiometric core-shell molecular imprinted quantum dot fluorescent coding microsphere, a preparation method and an application thereof. Background Art

[0002] As people's living standards improve, consumers pay more and more attention to food safety issues. As the most direct means to ensure food safety and consumer health, the development of rapid food safety detection methods has important practical significance. At this stage, how to further ensure the accuracy of detection methods and improve the sensitivity of detection are still two key issues in the development of food safety detection methods.

[0003] Fluorescence sensors are composed of materials with fluorescent properties. Such materials can interact with analytes and convert the concentration of the target into a fluorescent signal. The concentration of the target can be quantitatively analyzed according to the intensity of the change in the fluorescent signal. Combined with molecular imprinting technology, molecularly imprinted fluorescent sensors not only have the specific recognition ability of molecularly imprinted polymers (MIPs), but also show the advantages of highly sensitive fluorescence detection of fluorescent sensors. However, the currently developed molecularly imprinted fluorescent sensors are still mainly based on single emission fluorescence signals. Although the preparation process is relatively simple, they are easily interfered by various factors, such as instrument background interference, external environmental conditions, and interferents in the sample, which causes large errors in the measurement results. If you want to get accurate results, you can only strictly control the experimental conditions. Based on this, the researchers further proposed the concept of ratiometric fluorescence sensors, that is, using two fluorescent materials with different emission wavelengths to construct a fluorescence sensor with two fluorescent groups at the same time. When testing, the concentration of the target can be quantitatively calculated according to the ratio of the two different emission fluorescence intensities. This method can eliminate external interference through the ratio of the two signal intensities and improve the accuracy and sensitivity of the detection results.

[0004] In addition to the study of the output signal of the fluorescence sensor, the coding matrix is ​​also an important factor affecting the sensitivity and detection efficiency of the sensing system in the preparation of molecular imprinted fluorescent coded microspheres. In previous studies, traditional solid molecular imprinted polymers were mostly used as coding matrices. The resulting polymers were too cross-linked, resulting in low adsorption of the target, low mass transfer rate during adsorption, and too long time to reach adsorption equilibrium and detection, which is not conducive to its application in rapid detection. The core-shell molecular imprinted polymer prepared by surface imprinting technology as a coding matrix is ​​an effective way to solve this problem. In the preparation process, the core is used as a supporting material, and molecular imprinted polymers are prepared on its surface to ensure that the imprinted sites only exist in the external molecular imprinted shell. This structure is very beneficial for the adsorption and desorption of the target, which can effectively improve the mass transfer rate and shorten the coding time and detection time. The metal organic framework material ZIF-8 not only has a large specific surface area, strong adsorption, high porosity, simple preparation, but also has good biocompatibility and low toxicity. It is an ideal core material.

[0005] Norfloxacin (NOR) is a common fluoroquinolone drug, which is widely used to treat urinary, intestinal, respiratory and abdominal infections caused by Gram-positive and Gram-negative bacteria. Due to the abuse of NOR in animal husbandry and aquaculture, it is easy to cause its residues in animal products, endangering the health of consumers. Therefore, it is necessary to establish a rapid detection method with high accuracy and sensitivity for the rapid detection of NOR in food matrices. Summary of the invention

[0006] In order to solve the technical problems raised above, the present invention provides a ratio-type core-shell molecular imprinted quantum dot fluorescent coding microsphere, preparation method and application. The present invention uses metal organic framework material as a primary coding matrix and core-shell molecular imprinted polymer as a secondary coding matrix, combines secondary coding technology, and utilizes two ZnCdSe / ZnS quantum dots with different emission wavelengths to prepare ratio-type core-shell molecular imprinted quantum dot fluorescent coding microspheres, and the coding microspheres are used to detect trace amounts of norfloxacin in food, achieving highly selective quantitative detection of targets (such as NOR). The coding microspheres of the present invention are used in trace amounts of norfloxacin in food, which can not only effectively eliminate the errors and adverse effects of traditional detection methods, but also significantly improve the accuracy and reliability of detecting the target.

[0007] The ratiometric core-shell molecularly imprinted quantum dot fluorescent coding microspheres prepared by the present invention can not only increase the specific surface area, effectively improve the coding efficiency and fluorescence intensity, and shorten the detection time, but the metal organic framework material ZIF-8 core can also be used as a "reaction container" for loading one of the quantum dots, which can effectively realize the effective separation of the two quantum dots, avoid mutual interference in the fluorescence intensity of the two quantum dots, and ensure the accuracy of the detection system in practical applications.

[0008] The ratiometric core-shell molecularly imprinted quantum dot fluorescent coding microspheres provided by the present invention are mainly prepared by the following method:

[0009] The metal organic framework material was used as a primary encoding matrix, and then encoded with green quantum dots with an emission wavelength of 525 nm, and then used as a carrier to synthesize green core-shell molecular imprinted quantum dot fluorescent encoding microspheres on its surface;

[0010] Then, the green core-shell molecular imprinted quantum dot fluorescent coding microspheres were used as secondary coding matrix; the red quantum dots were dispersed in an organic solvent, the secondary coding matrix was added, ultrasonic mixing was performed and then oscillated to combine the red quantum dots with an emission wavelength of 625 nm and the green core-shell molecular imprinted quantum dot fluorescent coding microspheres to complete the secondary coding, and the supernatant was washed until there was no fluorescence, and dried to constant weight to obtain ratio-type core-shell molecular imprinted quantum dot fluorescent coding microspheres.

[0011] Preferably, the quantum dots with an emission wavelength of 525 nm are green ZnCdSe / ZnS quantum dots; the quantum dots with an emission wavelength of 625 nm are red ZnCdSe / ZnS quantum dots; and the organic solvent is n-hexane.

[0012] The above method specifically comprises the following steps:

[0013] (1) Preparation of metal organic framework material ZIF-8: ultrasonically dissolving zinc nitrate in methanol to obtain zinc nitrate solution; ultrasonically dissolving 2-methylimidazole in methanol to obtain 2-methylimidazole solution; slowly adding the 2-methylimidazole solution to the zinc nitrate solution, stirring at room temperature in the dark for 24 hours; wherein the molar ratio of zinc nitrate to 2-methylimidazole is 1:4, centrifuging, removing the supernatant and retaining the precipitate, washing the precipitate with methanol and vacuum drying to constant weight, to obtain the metal organic framework material ZIF-8;

[0014] (2) Preparation of ZIF-8 green quantum dot fluorescent coding material: using the metal organic framework material ZIF-8 obtained in (1) as a primary coding matrix, dispersing it in an organic solvent, adding green quantum dots, the mass ratio of the metal organic framework material ZIF-8 to the green quantum dots being 500:9, mixing, oscillating, completing primary coding, centrifuging to retain the precipitate, washing until there is no fluorescence in the supernatant, and drying to obtain the carrier ZIF-8 green quantum dot fluorescent coding material;

[0015] (3) Preparation of green core-shell molecular imprinted quantum dot fluorescent coding microspheres: Take the carrier ZIF-8 green quantum dot fluorescent coding material obtained in (2), add it to the porogen, after ultrasonic dispersion, add the template molecule, functional monomer, cross-linking agent, initiator, ultrasonicate, pass nitrogen to deoxygenate, seal in a water bath, wash with acetonitrile, filter, dry at 50-65°C for 20-30h, oscillate at a constant temperature, elute with methanol to remove the template molecule, and dry at 50-65°C for 20-30h to obtain green core-shell molecular imprinted quantum dot fluorescent coding microspheres;

[0016] (4) Preparation of ratiometric core-shell molecular imprinted quantum dot fluorescent coded microspheres: The green core-shell molecular imprinted quantum dot fluorescent coded microspheres in (3) are used as secondary coding matrix; red quantum dots are dispersed in an organic solvent, and the secondary coding matrix is ​​added, and the mixture is ultrasonically mixed and then oscillated to complete the secondary coding. The precipitate is retained by centrifugation, and washed with n-hexane until there is no fluorescence in the supernatant. The microspheres are dried in the dark at room temperature to a constant weight to obtain ratiometric core-shell molecular imprinted quantum dot fluorescent coded microspheres.

[0017] Preferably, the green quantum dots in (2) are green ZnCdSe / ZnS quantum dots with an emission wavelength of 525 nm; and the red quantum dots in (4) are red ZnCdSe / ZnS quantum dots with an emission wavelength of 625 nm.

[0018] Preferably, the green quantum dots in (2) are reference signals, and the red quantum dots in (4) are response signals.

[0019] Preferably, in (3), the porogen is acetonitrile; the template molecule is norfloxacin; the functional monomer is acrylamide; the crosslinking agent is composed of ethylene glycol dimethacrylate and divinylbenzene; and the initiator is azobisisobutyronitrile; wherein the molar ratio of ethylene glycol dimethacrylate to divinylbenzene is 1:1; the molar ratio of template molecule: functional monomer: crosslinking agent is 1:4:20; and the molar ratio of the functional monomer + crosslinking agent: initiator is 20:1.

[0020] Preferably, during drying in (3), the temperature is 60° C. and the drying time is 24 h.

[0021] Preferably, the organic solvent in (4) is n-hexane.

[0022] In the preparation process of ratiometric core-shell molecularly imprinted quantum dot fluorescent coding microspheres, the present invention uses the metal organic framework material ZIF-8 as the core and the NOR molecular imprinting layer as the shell layer to form a core-shell structure. The core-shell structure has the following beneficial effects: (1) From the perspective of improving the stability of quantum dots, the ZIF-8 material has a large specific surface area and adsorption capacity, and can be used as a "reaction container" for the effective loading of green quantum dots. The ZIF-8 material and the outer molecular imprinting shell layer both have a certain "protective" effect on the quantum dots, which can improve the stability of the quantum dots. Qualitative analysis; (2) From the perspective of effective separation of two different quantum dots, the two quantum dots are located in the core of the ZIF-8 material and the molecular imprinting shell, respectively. This effective separation can avoid the influence on the fluorescence performance of the quantum dots caused by the close distance; (3) From the perspective of improving the mass transfer rate, in this core-shell structure, the imprinting sites are concentrated on the surface of the molecular imprinting polymer (green core-shell molecular imprinting quantum dot fluorescent coding microspheres), which effectively solves the problems of traditional molecular imprinting polymers such as too deep embedding of imprinting sites, difficulty in elution of template molecules, and low mass transfer rate, thereby improving the encoding efficiency and shortening the detection time.

[0023] The ratiometric core-shell molecular imprinted quantum dot fluorescent coding microspheres prepared by the method of the present invention are combined with organic solvents to construct a ratiometric core-shell molecular imprinted quantum dot fluorescent coding sensing system, which is used to detect the NOR content in food and is also the scope of key protection of the present invention.

[0024] The above-mentioned food is any one of milk, pork, chicken, and fish. Of course, it can also be other types of food. The present invention is just an enumeration and not an exhaustive list.

[0025] In the present invention, a metal organic framework material is used as a primary coding matrix, a core-shell molecular imprinting polymer is used as a secondary coding matrix, and two ZnCdSe / ZnS quantum dots with different emission wavelengths are used as fluorescent signals. Combined with fluorescence coding technology, ratiometric core-shell molecular imprinting quantum dot fluorescent coding microspheres capable of specifically identifying NOR are prepared through secondary coding. The microspheres not only have a strong specific recognition function for NOR, but also have the excellent fluorescence properties of ZnCdSe / ZnS quantum dots.

[0026] In addition, the ratiometric core-shell molecular imprinted quantum dot fluorescent coding microspheres provided in the present invention are added to a solvent containing a sample to be tested (see Example 5 for details), and the obtained fluorescent sensor can be used for a rapid detection method for NOR with accurate quantitative analysis, which solves the problems of complicated detection conditions, long detection time and high professional operation requirements in the current NOR detection process. Moreover, due to the advantage of faster mass transfer rate of the core-shell structure, in the actual detection process of NOR, the detection time can be shortened to 15 minutes.

[0027] The beneficial effects of the present invention are:

[0028] (1) The present invention combines a core-shell molecular imprinted polymer with a large specific surface area, a fast mass transfer rate, and a high adsorption amount with two ZnCdSe / ZnS quantum dots with different emission wavelengths to prepare ratiometric core-shell molecular imprinted quantum dot fluorescent coding microspheres. The preparation process of the ratiometric fluorescence sensor is simplified by using a two-time encoding method, and the requirements for the performance of the quantum dots are weakened; the core-shell structure can avoid the mutual interference of the two quantum dots on the fluorescence performance, and give the material a larger specific surface area, accelerate the mass transfer rate, and shorten the detection time, so that the microspheres have the advantages of high selectivity, fast adsorption rate, low external interference, and high fluorescence quantum yield;

[0029] (2) The ratiometric core-shell molecular imprinted quantum dot fluorescent coding microspheres prepared by the present invention provide inherent built-in correction through two fluorescent signals, which can eliminate adverse effects to a certain extent, not only solving the problems existing in single-emission fluorescent sensors, but also improving detection accuracy and expanding the dynamic detection range;

[0030] (3) The ratiometric core-shell molecular imprinted quantum dot fluorescent coding microspheres constructed by the present invention are applied to the detection of trace amounts of norfloxacin in food. Not only is the detection time short, the fluorescent signal response is rapid, and the pre-treatment process is simple, it can achieve rapid identification and quantitative analysis of NOR in milk, chicken, pork, and fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Transmission electron microscopy images of (a) ZIF-8, (b) ZIF-8@G-QDs, (c) ZIF-8@G-QDs@MIPs, and (d) ZIF-8@G-QDs@MIPs@R-QDs encoded microspheres;

[0032] Figure 2 XRD patterns of ZIF-8, ZIF-8@G-QDs, ZIF-8@G-QDs@MIPs, and ZIF-8@G-QDs@MIPs@R-QDs prepared in Example 1;

[0033] Figure 3 The nitrogen adsorption-desorption isotherms of ZIF-8@G-QDs@MIPs@R-QDs in Example 1 and the MIPs prepared in Comparative Example 1;

[0034] Figure 4 The effect of different encoding time on the fluorescence intensity of ZIF-8@G-QDs@MIPs@R-QDs in Example 1 and MIPs fluorescence encoding microspheres in Comparative Example 1;

[0035] Figure 5Fluorescence intensity of (a) G-QDs quantum dots, (b) R-QDs quantum dots and (c) ZIF-8@G-QDs@MIPs@R-QDs fluorescent coding microspheres in different solvents;

[0036] Figure 6 is the temporal response of ZIF-8@G-QDs@MIPs@R-QDs microspheres to NOR detection;

[0037] Figure 7 This is the preparation process of ZIF-8@G-QDs@MIPs@R-QDs microspheres. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0039] Example 1

[0040] (1) 0.7344 g zinc nitrate and 0.8106 g 2-methylimidazole were dissolved in 50 mL methanol respectively, and the two were dissolved by ultrasound to obtain a zinc nitrate solution and a 2-methylimidazole solution. The 2-methylimidazole solution was slowly added to the zinc nitrate solution, and stirred for 24 h at room temperature in the dark. The obtained suspension was centrifuged at 8,000 rpm for 5 min to obtain a precipitate, and the precipitate was washed with methanol for 3 times, and dried in a vacuum oven at 40° C. for 8 h to obtain a metal organic framework material ZIF-8;

[0041] (2) Using the metal organic framework material ZIF-8 in (1) as a primary encoding matrix, 50 mg of the metal organic framework material ZIF-8 was added to a 2 mL centrifuge tube containing 1.5 mL of n-hexane, and the ZIF-8 was completely dispersed in the n-hexane by ultrasonication for 10 min. 300 μL of green quantum dots (G-QDs) were added. The green quantum dots were green ZnCdSe / ZnS quantum dots with an emission wavelength of 525 nm. The mixture was oscillated on an oscillator in the dark for 10 min to complete the primary encoding. The mixture was then centrifuged at 10,000 rpm for 10 min, the supernatant was removed, and the precipitate was washed with n-hexane until there was no fluorescence in the supernatant. The mixture was then dried in the dark at 25 °C to a constant weight. The carrier ZIF-8 green quantum dot fluorescent encoding material with G-QDs as the reference signal was obtained: ZIF-8@G-QDs.

[0042] (3) 50 mg of ZIF-8@G-QDs was added to a 50 mL round-bottom flask containing 20 mL of porogen acetonitrile, and the ZIF-8@G-QDs were completely dispersed in the acetonitrile by ultrasonication for 10 min. 0.1 mmol of template molecule NOR was added and dissolved by ultrasonication. 0.4 mmol of functional monomer acrylamide, 1 mmol of cross-linking agent ethylene glycol dimethacrylate, 1 mmol of cross-linking agent divinylbenzene and 20 mg of initiator azobisisobutyronitrile were added to the round-bottom flask, and ultrasonication was continued for 5 min. Nitrogen was introduced for 15 min to deoxygenate, and the flask was sealed under the protection of nitrogen. Polymerization was carried out in a water bath at 60 ° C. The obtained polymer was washed with acetonitrile, filtered, and dried in an oven at 60 ° C for 24 h. Then methanol was added to elute the material with a constant temperature oscillator. After removing the template molecule NOR, it was dried in an oven at 60 ° C for 24 h to obtain green core-shell molecularly imprinted quantum dot fluorescent coding microspheres: ZIF-8@G-QDs@MIPs;

[0043] (4) Select red ZnCdSe / ZnS quantum dots (R-QDs) with an emission wavelength of 625 nm as the fluorescent element of the response signal, use ZIF-8@G-QDs@MIPs as the secondary encoding matrix, perform secondary encoding, and prepare ratiometric core-shell molecular imprinted quantum dot fluorescent encoding microspheres. The specific steps are as follows:

[0044] 1 mL of n-hexane and 100 μL of 3 mg mL -1 The red quantum dot solution was added to a 2 mL centrifuge tube and ultrasonically mixed. Then, 10 mg of ZIF-8@G-QDs@MIPs was added and the ZIF-8@G-QDs@MIPs were evenly dispersed by ultrasound. The mixture was oscillated on an oscillator in the dark for 1 min to complete the secondary encoding. The mixture was then centrifuged at 10,000 rpm for 10 min, the supernatant was removed, and the precipitate was washed with n-hexane for 3 times until no fluorescence signal of the red ZnCdSe / ZnS quantum dots was detected in the supernatant. The mixture was then dried in the dark at 25 °C to constant weight to obtain ratiometric core-shell molecular imprinted quantum dot fluorescent coding microspheres: ZIF-8@G-QDs@MIPs@R-QDs.

[0045] In order to better understand the performance of the encoded microspheres provided by the present invention, we systematically characterized the prepared ZIF-8, ZIF-8@G-QDs, ZIF-8@G-QDs@MIPs and ZIF-8@G-QDs@MIPs@R-QDs, including analyzing their morphology and structure using transmission electron microscopy (TEM) and X-ray diffraction (XRD), and evaluating their specific surface area by nitrogen adsorption-desorption (BET) test. The results are shown in Figure 1-3 .

[0046] from Figure 1(a) It can be seen that the prepared metal organic framework material ZIF-8 presents a clear rhombic dodecahedron structure, which is consistent with previous research reports, indicating the successful preparation of the ZIF-8 material. After encoding with G-QDs fluorescent elements, ZIF-8 can effectively load G-QDs quantum dots to obtain ZIF-8@G-QDs. The presence of G-QDs quantum dots can be clearly seen on the ZIF-8@G-QDs composite material ( Figure 1 (b)), indicating that during the encoding process, ZIF-8 can be used as a "reaction container" to effectively load G-QDs. At the same time, the ZIF-8 material still maintains its original dodecahedral structure, indicating the successful preparation of the ZIF-8@G-QDs composite material.

[0047] Depend on Figure 1 (c) It can be seen that the obtained ZIF-8@G-QDs@MIPs presents an obvious core-shell structure, with the core being the metal organic framework material ZIF-8 and the outer layer being the NOR molecular imprinting layer. The formation of the core-shell structure can avoid the mutual interference of the fluorescence intensities of the two quantum dots and increase the stability of the fluorescence intensities of the two quantum dots. In addition, the core-shell structure can increase the specific surface area of ​​the microspheres, improve the mass transfer rate between the encoding matrix and the quantum dots, and shorten the encoding time.

[0048] Depend on Figure 1 As can be seen from (d) in the figure, after encoding ZIF-8@G-QDs@MIPs with R-QDs with an emission wavelength of 625 nm, the microspheres still maintain the core-shell structure before encoding, and the presence of R-QDs can be clearly observed on the molecular imprinting shell layer. These results indicate the successful preparation of the ratio-type core-shell molecular imprinting quantum dot fluorescent encoding microspheres in the present invention.

[0049] Depend on Figure 2 It can be seen that ZIF-8@G-QDs, ZIF-8@G-QDs@MIPs and encoded ZIF-8@G-QDs@MIPs@R-QDs all have the characteristic diffraction peaks of ZIF-8 materials, indicating that the materials contain ZIF-8 carriers, and that they contain ZIF-8 in the subsequent molecular imprinting synthesis and quantum dot encoding processes, indicating that the structure of ZIF-8 will not be destroyed in the subsequent operations, further proving the successful preparation of core-shell molecular imprinting polymers.

[0050] Depend on Figure 3 It can be seen that according to the Brunauer-Emmett-Teller (BET) theoretical calculation, the specific surface area of ​​the ZIF-8@G-QDs@MIPs@R-QDs encoded microspheres prepared in the present invention is 203.51 m 2 ·g -1 .

[0051] Example 2

[0052] Based on Example 1, the secondary encoding time of ZIF-8@G-QDs@MIPs@R-QDs fluorescent encoding microspheres was optimized.

[0053] 10 mg of ZIF-8@G-QDs@MIPs was added to 1 mL of n-hexane for ultrasonic dispersion, and then 100 μL of red ZnCdSe / ZnS quantum dots was added. After oscillation in an oscillator for 1, 3, 5, 10, 15, and 20 min, the mixture was centrifuged at 10,000 rpm for 10 min. The supernatant was removed, and n-hexane was added to wash the precipitate three times until there was no fluorescence signal of ZnCdSe / ZnS quantum dots in the supernatant. The mixture was dried at 25 °C in the dark to constant weight. The ZIF-8@G-QDs@MIPs@R-QDs coded microspheres with different coding times were dispersed in acetonitrile (concentration of 1 mg mL -1 ), and then the fluorescence intensity (E x =365nm, E m =625nm), and the relationship between the fluorescence intensity of the coded microspheres and the secondary coding time was analyzed. The experimental results are shown in Figure 4 .

[0054] from Figure 4 It can be seen that ZIF-8@G-QDs@MIPs as a secondary coding matrix can achieve encoding with red quantum dots R-QDs in a relatively short time. When the coding time is 1 min, the fluorescence intensity is close to the adsorption equilibrium. As the coding time increases, the fluorescence intensity increases significantly. In order to shorten the coding time and improve the coding efficiency, 1 min is selected as the final coding time.

[0055] Example 3

[0056] Based on Example 1, the stability of the fluorescence properties of the obtained ZIF-8@G-QDs@MIPs@R-QDs fluorescent encoded microspheres in different polar organic solvents was evaluated.

[0057] The ZIF-8@G-QDs@MIPs@R-QDs fluorescent coded microspheres were added to solvents of different polarities (methanol, acetonitrile, petroleum ether, chloroform, toluene, and n-hexane) and ultrasonically mixed for 10 min. The final concentration of the coded microspheres was 1 mg mL -1 The fluorescence intensity of the encoded microspheres (E x =365nm, E m =525nm, E m =625nm), the results are shown in Table 1 and Figure 5 shown.

[0058] Table 1 Fluorescence emission wavelength and fluorescence intensity of fluorescent coded microspheres in different solvents

[0059]

[0060] Table 1 and Figure 5 The results show that G-QDs after primary encoding with ZIF-8 and R-QDs after secondary encoding with ZIF-8@G-QDs@MIPs dispersed in solvents of different polarities both exhibit bright characteristic fluorescence emission peaks. The emission peaks are symmetrically distributed and do not undergo obvious displacement, proving that the core-shell structure can avoid the interference of solvents of different polarities on R-QDs and enhance the stability of quantum dots in strong polar solvents.

[0061] Example 4

[0062] Based on Example 1, the response time of ZIF-8@G-QDs@MIPs@R-QDs fluorescently encoded microspheres to the target NOR was optimized.

[0063] The ZIF-8@G-QDs@MIPs@R-QDs fluorescently encoded microspheres were dispersed in acetonitrile (concentration 1 mg mL -1 ), then add NOR standard solution to make the concentration of NOR in the coded microsphere solution 400 μg·L -1 The samples were placed in an oscillator and oscillated for 1, 3, 5, 10, 15, 20, and 30 minutes respectively. The fluorescence intensity was measured using a fluorescence spectrophotometer to evaluate the effect of different response times on the fluorescence intensity of the encoded microspheres. The results are shown in Figure 6 shown.

[0064] from Figure 6 It can be seen from the results that after adding NOR, the fluorescence quenching degree of ZIF-8@G-QDs@MIPs@R-QDs microspheres changed rapidly and reached equilibrium within 15 minutes. After extending the response time, the fluorescence quenching degree of the encoded microspheres did not increase significantly. Therefore, the optimal response time of the fluorescent encoded microspheres to NOR was reached at 15 minutes. Compared with the detection time of solid molecular imprinting in previous work (patent number ZL 2022 1 0125204.2), the detection time in the present invention is shortened by half. The main reason is that the unique core-shell structure concentrates the specific recognition sites on the surface of the encoded microspheres, which can increase the mass transfer rate between the encoding matrix and the quantum dots, and NOR is more likely to bind to the recognition sites, thereby shortening the detection time.

[0065] Example 5

[0066] A NOR fluorescence sensor was constructed using ratiometric core-shell molecular imprinted quantum dot fluorescence coding microspheres for the detection of NOR in milk, chicken, pork and fish. The feasibility of the fluorescence sensor in actual sample detection was evaluated through spike recovery experiments.

[0067] Weigh 1g or 1mL of the sample to be tested and add it to 10mL of acetonitrile. After shaking for 10min, centrifuge at 7000rpm for 5min to collect the supernatant. Repeat the extraction of the residue obtained by centrifugation three times, combine the supernatants, add 10mL of acetonitrile saturated n-hexane solution, shake for 1min to remove the fat in the extract, evaporate it to near dryness, and redissolve it in 1mL of acetonitrile as the test solution. Add ZIF-8@G-QDs@MIPs@R-QDs fluorescent coded microspheres to the test solution, disperse it ultrasonically, and obtain a fluorescent sensor. The final concentration of the sensor is 1mg·mL -1 , shaken in the dark at room temperature for 15 minutes. After sufficient reaction, the fluorescence intensity of each group of sample solutions was tested using a fluorescence spectrophotometer. The results are shown in Table 2.

[0068] Table 2 Recovery and RSD values ​​of NOR detection in different samples

[0069]

[0070]

[0071] Through the spike recovery experiment, the sensor was used to determine the recovery rate of NOR in four food samples to verify the accuracy of the sensor in actual samples. After measurement, the constructed fluorescent sensor was successfully applied to the trace detection of NOR in milk, chicken, pork, and fish. The recovery rate of the spiked sample was 89.76%-103.21%, and the relative standard deviation (RSD) was 1.58%-8.96%. The above results show that the prepared ZIF-8@G-QDs@MIPs@R-QDs fluorescent sensor is feasible for the detection of NOR in the spiked sample and can meet the detection of NOR in complex food samples. And the microspheres of the present invention are applied to the detection of norfloxacin in food, which can shorten the entire detection process to 15 minutes, with short detection time and high efficiency.

[0072] In summary, the present invention utilizes core-shell molecular imprinting polymers and ZnCdSe / ZnS quantum dots, combined with optical coding technology, to develop an efficient and accurate ratiometric fluorescence sensor (fluorescent coding microspheres), which can achieve rapid detection of NOR and quantitative analysis of residues in a variety of foods, significantly improving the efficiency and reliability of food safety testing.

[0073] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

[0074] Comparative Example 1

[0075] On the basis of Example 1, without adding metal organic framework ZIF-8 and G-QDs quantum dots, traditional molecular imprinted polymers (MIPs) and corresponding fluorescent encoded microspheres (MIPs@R-QDs) were prepared to demonstrate the advantages of core-shell molecular imprinted polymers and fluorescent encoded microspheres.

[0076] By N 2 Adsorption-desorption analysis was used to characterize the MIPs prepared in this comparative example. The characterization results are shown in Figure 3 .

[0077] like Figure 3 As shown in Figure 2, according to the Brunauer-Emmett-Teller (BET) theory, the specific surface area of ​​MIPs is 4.5 m 2 ·g -1 .

[0078] Compared with the specific surface area of ​​ZIF-8@G-QDs@MIPs in Example 1, the specific surface area of ​​ZIF-8@G-QDs@MIPs is significantly higher than that of MIPs. The high specific surface area is beneficial to increasing the adsorption amount of template molecules by the encoded microspheres, and can promote more R-QDs to enter the polymer microspheres during encoding, thereby increasing the capacity of the encoded quantum dots.

[0079] Comparative Example 2

[0080] On the basis of Example 2, the conventional molecular imprinting polymer (MIPs) was encoded in the same way and oscillated for 1, 3, 5, 10, 15, 20, 25, and 30 min, respectively, to prepare fluorescent encoded microspheres MIPs@R-QDs. The fluorescence intensity (E x =365nm, E m =625nm), analyze the relationship between fluorescence intensity and oscillation encoding time, and the experimental results are shown in Figure 4 .

[0081] Combination Figure 4 From the results, it can be seen that the adsorption equilibrium is reached after 25 minutes of encoding of MIPs and R-QDs. The fluorescence intensity does not increase significantly after the encoding time is prolonged. Compared with the encoding time of ZIF-8@G-QDs@MIPs@R-QDs fluorescent encoding microspheres in Example 2, the time to reach the adsorption equilibrium is longer. The main reason is that the unique core-shell structure can increase the mass transfer rate between the encoding matrix and the quantum dots, thereby improving the encoding efficiency and shortening the encoding time.

[0082] Comparative Example 3

[0083] Based on Example 3, pure G-QDs solution and pure R-QDs solution were added to solvents of different polarities (methanol, acetonitrile, petroleum ether, chloroform, toluene, and n-hexane), respectively, and the fluorescence intensity (E x =365nm, E m =525nm, E m =625nm), the results are shown in Table 3 and Figure 5 shown.

[0084] Table 3 Fluorescence emission wavelength and fluorescence intensity of G-QDs and R-QDs quantum dots in different solvents

[0085]

[0086] From Table 3 and Figure 5 The results show that the fluorescence intensity of G-QDs quantum dots and R-QDs quantum dots in strong polar solvents (methanol, acetonitrile) is significantly reduced, and the emission peak of R-QDs quantum dots is red-shifted by 2-3nm. Compared with the results in Table 1, the encoded microspheres can still maintain good fluorescence intensity in strong polar solvents, proving that the ZIF-8 material and the outer molecular imprinting shell have a certain "protective" effect on G-QDs. The encoding of R-QDs into the molecular imprinting shell can improve the stability of quantum dots in polar solvents. This unique structure and encoding method can improve the stability of quantum dots in strong polar solvents. When the encoded microspheres are used for NOR detection in polar solvents, they can stably output fluorescence signals to ensure the stability of the detection results.

Claims

1. A ratiometric core-shell molecularly imprinted quantum dot fluorescent coding microsphere, characterized in that: The microspheres are mainly prepared by the following method: The metal organic framework material was used as a primary encoding matrix, and then encoded with green quantum dots with an emission wavelength of 525 nm. After that, it was used as a carrier to synthesize green core-shell molecular imprinted quantum dot fluorescent encoding microspheres on its surface. Then, the green core-shell molecular imprinted quantum dot fluorescent coding microspheres were used as the secondary coding matrix; the red quantum dots were dispersed in an organic solvent, the secondary coding matrix was added, ultrasonic mixing was performed and then oscillated to allow the red quantum dots with an emission wavelength of 625 nm and the green core-shell molecular imprinted quantum dot fluorescent coding microspheres to combine to complete the secondary coding, and the supernatant was washed until there was no fluorescence, and dried to a constant weight to obtain ratio-type core-shell molecular imprinted quantum dot fluorescent coding microspheres.

2. A ratiometric core-shell molecularly imprinted quantum dot fluorescent coding microsphere according to claim 1, characterized in that: The quantum dots with an emission wavelength of 525 nm are green ZnCdSe / ZnS quantum dots; The quantum dots with an emission wavelength of 625 nm are red ZnCdSe / ZnS quantum dots; The organic solvent is n-hexane.

3. The method for preparing ratiometric core-shell molecularly imprinted quantum dot fluorescent coding microspheres according to claim 1, comprising the following steps: (1) Preparation of metal organic framework material ZIF-8: ultrasonically dissolving zinc nitrate in methanol to obtain a zinc nitrate solution; ultrasonically dissolving 2-methylimidazole in methanol to obtain a 2-methylimidazole solution; slowly adding the 2-methylimidazole solution to the zinc nitrate solution, and stirring at room temperature in the dark for 24 hours; wherein, The molar ratio of zinc nitrate to 2-methylimidazole was 1:4, centrifuged, the supernatant was removed and the precipitate was retained, the precipitate was washed with methanol and then vacuum dried to constant weight to obtain the metal organic framework material ZIF-8; (2) Preparation of ZIF-8 green quantum dot fluorescent coding material: The metal organic framework material ZIF-8 obtained in (1) is used as a primary coding matrix, dispersed in an organic solvent, and green quantum dots are added. The mass ratio of the metal organic framework material ZIF-8 to the green quantum dots is 500:

9. The material is mixed and shaken to complete the primary coding. The precipitate is retained by centrifugation and washed until there is no fluorescence in the supernatant. The material is dried to obtain the carrier ZIF-8 green quantum dot fluorescent coding material. (3) Preparation of green core-shell molecular imprinted quantum dot fluorescent coding microspheres: Take the carrier ZIF-8 green quantum dot fluorescent coding material obtained in (2), add it to the porogen, after ultrasonic dispersion, add the template molecule, functional monomer, cross-linking agent, initiator, ultrasonicate, pass nitrogen to deoxygenate, seal in a water bath, wash with acetonitrile, filter, dry at 50-65°C for 20-30 h, oscillate at a constant temperature, elute with methanol to remove the template molecule, and dry at 50-65°C for 20-30 h to obtain green core-shell molecular imprinted quantum dot fluorescent coding microspheres; (4) Preparation of ratiometric core-shell molecular imprinted quantum dot fluorescent coded microspheres: The green core-shell molecular imprinted quantum dot fluorescent coded microspheres in (3) are used as the secondary coding matrix; the red quantum dots are dispersed in an organic solvent, and the secondary coding matrix is ​​added. The mixture is ultrasonically mixed and then oscillated to complete the secondary coding. The precipitate is retained by centrifugation and washed with n-hexane until there is no fluorescence in the supernatant. The mixture is dried in the dark at room temperature to a constant weight to obtain ratiometric core-shell molecular imprinted quantum dot fluorescent coded microspheres.

4. The method for preparing ratiometric core-shell molecularly imprinted quantum dot fluorescent coding microspheres according to claim 3, characterized in that: The green quantum dots described in (2) are green ZnCdSe / ZnS quantum dots with an emission wavelength of 525 nm; the red quantum dots described in (4) are red ZnCdSe / ZnS quantum dots with an emission wavelength of 625 nm.

5. The method for preparing ratiometric core-shell molecularly imprinted quantum dot fluorescent coding microspheres according to claim 3, characterized in that: The green quantum dots described in (2) are reference signals, and the red quantum dots described in (4) are response signals.

6. The method for preparing ratiometric core-shell molecularly imprinted quantum dot fluorescent coding microspheres according to claim 3, characterized in that: The porogen described in (3) is acetonitrile; The template molecule is norfloxacin; The functional monomer is acrylamide; The initiator is azobisisobutyronitrile; The crosslinking agent is composed of ethylene glycol dimethacrylate and divinyl benzene, wherein the molar ratio of ethylene glycol dimethacrylate to divinyl benzene is 1:1; The molar ratio of the template molecule: functional monomer: cross-linking agent is 1:4:20; The molar ratio of the functional monomer+crosslinking agent:initiator is 20:

1.

7. The method for preparing ratiometric core-shell molecularly imprinted quantum dot fluorescent coding microspheres according to claim 3, characterized in that: (3) During medium drying, the temperature is 60°C and the time is 24 h.

8. Use of the ratiometric core-shell molecularly imprinted quantum dot fluorescent coding microspheres as claimed in claim 1 in detecting trace amounts of norfloxacin in food.

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