WO2024169486A1 - 一种复合磁助比率荧光探针及其制备方法和应用 - Google Patents

一种复合磁助比率荧光探针及其制备方法和应用 Download PDF

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WO2024169486A1
WO2024169486A1 PCT/CN2024/072230 CN2024072230W WO2024169486A1 WO 2024169486 A1 WO2024169486 A1 WO 2024169486A1 CN 2024072230 W CN2024072230 W CN 2024072230W WO 2024169486 A1 WO2024169486 A1 WO 2024169486A1
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fluorescent probe
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孙宗保
李晨
邹小波
张文
高云龙
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Jiangsu University
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Definitions

  • the invention belongs to the technical field of nano material and sensor preparation, and in particular relates to a composite magnetic-assisted ratio fluorescent probe and a preparation method and application thereof.
  • Chloramphenicol is an effective broad-spectrum antibiotic that works by inhibiting bacterial protein synthesis.
  • excessive use and residual chloramphenicol can affect human health.
  • excessive chloramphenicol can cause irreversible damage to the hematopoietic system and digestive system; on the other hand, drug residues in food can also seriously threaten human health. Therefore, chloramphenicol has been banned in the global animal husbandry industry.
  • commonly used chloramphenicol detection and classification methods include enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC) and immunoassay.
  • the present invention provides a composite magnetic-assisted ratio fluorescent probe and a preparation method and application thereof;
  • the composite magnetic-assisted ratio fluorescent probe of the present invention is obtained by compounding a fluorescent microsphere probe and a magnetically functionalized europium polymer, and with the addition of chloramphenicol, the change in the fluorescence signal of the composite magnetic-assisted ratio fluorescent probe can be observed;
  • the composite magnetic-assisted ratio fluorescent probe has a built-in calibration function, which can reduce the interference caused by the environment and instruments, and has good selectivity for chloramphenicol, and has good anti-interference performance for common antibiotics, physiological substances, and inorganic ions, and can be used for visual detection of chloramphenicol in food.
  • the present invention first provides a composite magnetic-assisted ratio fluorescent probe, which is obtained by compounding a fluorescent microsphere probe and a magnetically functionalized europium polymer; the fluorescent microsphere probe is close in shape to a sphere and has a rough surface; the magnetically functionalized europium polymer is prepared by self-assembly, has a close in shape to a sphere and has an obvious layered structure; the fluorescent microsphere probe is evenly distributed on different layers of the magnetically functionalized europium polymer.
  • the present invention also provides a method for preparing the composite magnetic-assisted ratio fluorescent probe, which specifically comprises the following steps:
  • the citric acid and polyethyleneimine (PEI) are ultrasonically mixed and then reacted under constant temperature heating conditions 1. After the reaction 1 is completed, the mixture is cooled, eluted, purified, and dried to obtain BCDs. CaCl 2 , carboxymethyl cellulose (CMC) and BCDs are uniformly mixed, and then a Na 2 CO 3 aqueous solution is added to carry out a mineralization reaction 2. After the mineralization reaction 2 is completed, the mixture is centrifuged, washed, and dried to obtain BCDs@CaCO 3 .
  • BCDs@CaCO 3 N-hydroxysulfosuccinimide (NHS), carbodiimide hydrochloride (EDC), and chloramphenicol aptamer (CAP-Apt) were dispersed in MES buffer and mixed to perform reaction 3. After reaction 3, the mixture was centrifuged and washed to finally obtain the BCDs@CaCO 3 -Apt fluorescent probe.
  • NHS N-hydroxysulfosuccinimide
  • EDC carbodiimide hydrochloride
  • CAP-Apt chloramphenicol aptamer
  • the mass ratio of citric acid to PEI is 2:1; the BCDs reaction 1 is carried out at 200-250° C. for 6-10 hours;
  • the mass ratio of CaCl 2 , CMC, BCDs and Na 2 CO 3 is 1:1:1.5:1, and the condition of the mineralization reaction 2 is to react at room temperature for 24 to 30 hours.
  • the mass ratio of the BCDs@CaCO 3 , NHS and EDC is 5-15:2-8:3-12, the reaction time of reaction 3 is 4-10 hours, and the final concentration of CAP-Apt in the mixed solution is 0.1-1 mg/mL.
  • the mass ratio of ferric chloride hexahydrate, sodium citrate and sodium acetate is 3:1:6, and the addition ratio of ferric chloride hexahydrate to ethylene glycol is 0.3 g:10 mL; the condition of reaction 4 is to react at 200-250° C. for 10-16 hours;
  • Fe 3 O 4 is mixed with ethanol aqueous solution in a ratio of 1:5, the volume fraction of ethanol in the ethanol aqueous solution is 80%, the volume ratio of ammonia water and tetraethyl orthosilicate is 1:2, and the stirring reaction time is 12 to 15 hours.
  • TTA benzoyltrifluoroacetone
  • bpm 2,2'-bipyrimidine
  • the mass ratio of europium chloride hexahydrate ethanol solution, benzoyltrifluoroacetone (TTA), and 2,2'-bipyrimidine (bpm) is 2-8:5-10:1-4;
  • the total volume of ethanol in the mixed reaction system is equal to the total volume of europium chloride hexahydrate ethanol solution, benzoyl trifluoroacetone and 2,2'- The volume of bipyrimidine and the same;
  • the reaction time of the aging reaction 6 is 1 to 4 hours.
  • Chloramphenicol hapten (CAP-Hapten), N-hydroxysulfosuccinimide (NHS) and carbodiimide hydrochloride (EDC) were mixed in a methanol solution, and Eu-Fe 3 O 4 @SiO 2 -NH 2 in MES buffer was added and stirred for reaction for 9, separated and washed to finally obtain a magnetically functionalized europium coordination polymer, which was recorded as Eu-Fe 3 O 4 @SiO 2 -Hapten.
  • CAP-Hapten N-hydroxysulfosuccinimide
  • EDC carbodiimide hydrochloride
  • the dosage ratio of Fe 3 O 4 @SiO 2, N,N-dimethylformamide (DMF), [Eu(TTA) 3 ] 2 bpm, ammonia water and TEOS is 20-60 mg: 80-240 mL: 10-30 mg: 1.25-3.75 mL: 1-5 mL;
  • reaction 7 The conditions of reaction 7 are ultrasonic stirring for 10 to 20 minutes and reaction at room temperature for 10 to 15 hours;
  • the solvent of the 3-aminopropyltriethoxysilane (APTES) solution is anhydrous ethanol
  • the solvent of the Eu-Fe 3 O 4 @SiO 2 solution is pure water
  • the volume ratio of the 3-aminopropyltriethoxysilane (APTES) solution to the Eu-Fe 3 O 4 @SiO 2 solution is 10-50:1-5;
  • reaction 8 The conditions for reaction 8 are room temperature for 10 to 15 hours;
  • the mass ratio of the chloramphenicol hapten (CAP-Hapten), N-hydroxysulfosuccinimide (NHS), carbodiimide hydrochloride (EDC) and Eu-Fe 3 O 4 @SiO 2 is 1-5:2-10:3-15:10-50;
  • the conditions of the reaction 9 are stirring the reaction for 4 to 8 hours.
  • the BCDs@CaCO 3 -Apt solution and the Eu-Fe 3 O 4 @SiO 2 -Hapten solution were evenly mixed to obtain a composite magnetically assisted ratio fluorescent probe solution, which was recorded as BCDs@CaCO 3 -Apt/Eu-Fe 3 O 4 @SiO 2 -Hapten.
  • the volume ratio of the BCDs@CaCO 3 -Apt solution to the Eu-Fe 3 O 4 @SiO 2 -Hapten solution is 1-6:1-9;
  • the concentration of the BCDs@CaCO 3 -Apt solution is 10 mg/mL, and the concentration of the Eu-Fe 3 O 4 @SiO 2 -Hapten solution is 10 mg/mL, that is, the concentration of the BCDs@CaCO 3 -Apt solution is the same as the concentration of the Eu-Fe 3 O 4 @SiO 2 -Hapten solution.
  • the present invention also provides the use of the composite magnetic-assisted ratio fluorescent probe in visual detection of chloramphenicol, wherein the visual detection is semi-quantitative detection or quantitative detection.
  • the chloramphenicol aqueous solution was mixed with BCDs@CaCO 3 -Apt/Eu-Fe 3 O 4 @SiO 2 -Hapten to obtain a fluorescent composite system.
  • the standard curve of chloramphenicol concentration was fitted by fluorescence analysis, and the color of the fluorescent composite system under ultraviolet light was recorded.
  • the present invention has the following beneficial effects:
  • the composite magnetic ratio fluorescent probe of the present invention is detected by competition method, and the chloramphenicol hapten is used to compete with chloramphenicol for chloramphenicol aptamer for detection.
  • the chloramphenicol hapten can bind to the aptamer, but the binding ability is weaker than that of chloramphenicol; the aptamer has strong specificity and has high stability and environmental adaptability compared with antibodies and enzymes.
  • the detection time of the method of the present invention is shortened by half compared with other fluorescence analysis methods, and the detection limit is accurate from 0.7ng/mL of the existing detection method to 0.09ng/mL.
  • the composite magnetic-assisted ratio fluorescence probe of the present invention can observe two fluorescence signals. With the addition of chloramphenicol, one of the fluorescence signals will change. Due to the immune response, BCDs@CaCO 3 -Apt preferentially binds to chloramphenicol. Under the action of the magnetic field, the magnetically functionalized europium polymer is retained, and the blue fluorescence is removed after washing, so the blue fluorescence is weakened and the red fluorescence remains unchanged. Compared with the single fluorescence signal probe, the advantage of the present invention is that it can reduce the interference caused by the environment and instruments.
  • the composite magnetic-assisted ratio fluorescent probe of the present invention can produce gradient color changes under ultraviolet light after being mixed with chloramphenicol of different concentrations. As the concentration of chloramphenicol increases, the fluorescent color of the mixed system changes. The concentration range of chloramphenicol can be semi-quantitatively determined based on the color, making it easier to achieve visual detection of the object to be tested.
  • the composite magnetic-assisted ratio fluorescent probe of the present invention has good selectivity for chloramphenicol and good anti-interference performance for common antibiotics and inorganic ions, and has certain practical value in food detection.
  • the magnetically functionalized europium coordination polymer of the present invention introduces another ligand, 2,2'-bipyrimidine (bpm), into the binary europium complex to form a ternary complex, which greatly improves the luminescence intensity of the original complex.
  • the luminescence properties of europium ions are enhanced and modified under the action of the dual ligands, thereby improving the luminescence efficiency of the rare earth complex.
  • the ratio fluorescence formed by combining carbon dots and europium polymers in the present invention has a self-calibration function, which can eliminate fluctuations caused by external factors and improve the reliability and reproducibility of the detection results.
  • FIG. 1 is a transmission electron microscope image (A) and a laser confocal image (B) of BCDs@CaCO 3 -Apt obtained in the present invention, wherein 1-8 are single-sphere fluorescence confocal images at different levels.
  • FIG. 2 is a transmission electron microscope image (A) and a laser confocal image (B) of Eu-Fe 3 O 4 @SiO 2 -Hapten obtained in the present invention
  • FIGS. 1-4 are single-sphere fluorescence confocal images at different levels, respectively.
  • FIG3 is a fluorescence spectrum of the composite magnetic-assisted ratiometric fluorescent probe after adding different concentrations of chloramphenicol.
  • FIG4 is a calibration curve of the fluorescence intensity ratio F 435 /F 615 and the chloramphenicol concentration.
  • FIG5 is a fluorescence color diagram of the ratio fluorescence probe corresponding to different concentrations of chloramphenicol.
  • Figure 6 is a schematic diagram of the selectivity of the ratio fluorescent probe obtained by the present invention.
  • A is a schematic diagram of the selectivity of the composite magnetic-assisted ratio fluorescent probe for different types of antibiotics (cephapirin, amoxicillin, tetracycline, enrofloxacin and sulfamethoxazole)
  • B is a schematic diagram of the selectivity of the composite magnetic-assisted ratio fluorescent probe for different types of inorganic ions (potassium ions, sodium ions, magnesium ions, zinc ions, carbonate ions, sulfate ions, and nitrate ions).
  • 100 ⁇ L CAP-Apt 5.0 mg/mL, purchased from Shanghai Shenggong Biological Reagent Co., Ltd. was added and reacted for another 4 h.
  • the mixture was centrifuged at 5000 rpm for 10 min and washed with pure water for 3 times to finally obtain a BCDs@CaCO 3 -Apt fluorescent probe solution.
  • the prepared BCDs@CaCO 3 -Apt has a uniform size distribution without aggregation, and the particle size is 675 to 925 nm.
  • the laser confocal image shows that the fluorescence distribution is uniform.
  • FIG. 2 is a transmission electron microscope image (A) and a laser confocal microscope image (B) of Eu-Fe 3 O 4 @SiO 2 -Hapten; as shown in the transmission electron microscope image, the europium coordination polymer prepared by self-assembly has a clear layered structure, and the laser confocal microscope image shows that each layer has strong fluorescence.
  • 80mg Eu-Fe 3 O 4 @SiO 2 was dispersed in 50mL ultrapure water, and 8mL APTES was added, and the mixture was reacted on a shaker for 10h, and then the mixture was washed with ethanol and ultrapure water for 3 times respectively, and then vacuum dried at 50°C for 8h.
  • 8.0 mg CAP-Hapten, 40 mg NHS and 80 mg EDC were dissolved in 1 mL methanol and reacted for 4 h.
  • the mixture was added into MES buffer (5 mL) containing 100.0 mg Eu-Fe 3 O 4 @SiO 2 and stirred for 8 h. After washing, a magnetically functionalized europium coordination polymer was obtained, which was recorded as Eu-Fe 3 O 4 @SiO 2 -Hapten.
  • a chloramphenicol standard solution was prepared with PBS, and 100 ⁇ L of the chloramphenicol aqueous solution was mixed with the composite magnetic-assisted ratiometric fluorescent probe BCDs@CaCO 3 -Apt/Eu-Fe 3 O 4 @SiO 2 -Hapten described in Example 1 to obtain composite systems with chloramphenicol concentrations of 0, 0.1, 0.5, 1, 3, 5, 7, 10, 15 and 20 ng/mL, respectively.
  • the mixture was incubated at room temperature for 10 min, the supernatant of the mixture was removed, and the precipitate was collected by a magnet and then dispersed in 1 mL of PBS. Under 365 nm excitation light, the fluorescence spectra of the composite systems at different chloramphenicol concentrations were recorded at 350-650 nm.
  • the composite magnetic-assisted ratiometric fluorescent probe when there is no chloramphenicol, the composite magnetic-assisted ratiometric fluorescent probe emits red fluorescence and blue fluorescence simultaneously. With the addition of chloramphenicol, the fluorescence signal change can be observed. Due to the immune reaction, BCDs@CaCO 3 -Apt preferentially binds to chloramphenicol. Under the action of the magnetic field, the magnetically functionalized red fluorescence is retained, and the blue fluorescence is removed after washing, so the blue fluorescence is weakened.
  • the composite magnetic-assisted ratio fluorescent probe obtained in Example 1 was ratiometrically fluorescently compounded at different chloramphenicol concentrations, and the supernatant was removed after incubation for 10 minutes. The precipitate was then dispersed in PBS and placed under ultraviolet light. The color information corresponding to the above solution was recorded, and the color signal was corresponded to the chloramphenicol concentration respectively, and the color changes were recorded in order from small to large.
  • Figure 5 is a calibration curve of the fluorescence intensity ratio F435 / F615 and the chloramphenicol concentration. It can be seen from the figure that under 365nm ultraviolet light, it can be clearly seen by the naked eye that after adding different concentrations of chloramphenicol (0-20ng/mL), the fluorescence color of the mixed system changes from purple to red. Therefore, the composite magnetic-assisted ratio fluorescent probe of the present invention can realize semi-quantitative visual detection of chloramphenicol based on color information.
  • Example 6 Detection and analysis of chloramphenicol in milk samples
  • this embodiment selects the sample to be tested for residual detection, and the specific steps are as follows:
  • the above solution was placed in a fluorescence spectrometer, and the fluorescence spectrum was measured under 360 nm excitation light to obtain the fluorescence intensity ratio F 435 /F 615 , and the obtained F 435 /F 615 was brought into the fitting equation described in Example 4 to obtain the chloramphenicol content in the sample.
  • the accuracy of the composite magnetic-assisted ratio fluorescent probe in chloramphenicol detection was explored by the standard spike method, and the results showed that spiked chloramphenicol could be accurately measured, with a high recovery rate (from 97% to 107%) and good precision (RSD ⁇ 5.2%).
  • antibiotics cephapirin, amoxicillin, tetracycline, enrofloxacin and sulfamethoxazole
  • inorganic ions potassium ions, sodium ions, magnesium ions, zinc ions, carbonate ions, sulfate ions, nitrate ions
  • FIG6A is a schematic diagram of the selectivity of the composite magnetic-assisted ratiometric fluorescent probe obtained in the present invention for five antibiotics, namely, cefpiroximate, amoxicillin, tetracycline, enrofloxacin and sulfamethoxazole. It can be seen from the figure that the change in fluorescence ratio is only found in the solution containing CAP but not other spiked samples, indicating that the aptamer recognition has excellent selectivity, and also indicating that the composite system has specificity in detecting chloramphenicol and will not be interfered by other coexisting antibiotics.
  • five antibiotics namely, cefpiroximate, amoxicillin, tetracycline, enrofloxacin and sulfamethoxazole.
  • Chloramphenicol was mixed with different inorganic ions, and the mixed solutions were added to the composite ratio fluorescent probe solution described in Example 1 to obtain a composite system.
  • the concentration of inorganic ions in the final composite system was 500 ⁇ M, and the concentration of chloramphenicol was 50 ⁇ M.
  • the method described in Example 5 was used for detection, and the detection results are shown in Figure 6B.
  • Figure 6B is a schematic diagram of the selectivity of the composite ratio fluorescent probe for potassium ions, sodium ions, magnesium ions, zinc ions, carbonate ions, sulfate ions, and nitrate ions. It can be seen from the figure that no significant change was observed between the signal of the mixed solution and the signal when chloramphenicol exists alone, indicating that the sensor has good selectivity for chloramphenicol and will not be interfered by other ions in the solution.

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Abstract

本发明提供了一种复合磁助比率荧光探针及其制备方法和应用，属于纳米材料和传感器制备技术领域；本发明所述复合磁助比率荧光探针由蓝色荧光碳点和磁功能化的铕聚合物复合得到，随着氯霉素的加入可观察到复合磁助比率荧光探针的荧光信号变化；所述复合磁助比率荧光探针具有内置校准功能，能降低环境和仪器带来的干扰，并且其对氯霉素有良好的选择性，对常见的抗生素和无机离子具有良好的抗干扰性能，能够用于可视化检测食品中的氯霉素。

Description

一种复合磁助比率荧光探针及其制备方法和应用 技术领域
本发明属于纳米材料和传感器制备技术领域，具体涉及一种复合磁助比率荧光探针及其制备方法和应用。
背景技术
氯霉素(CAP)是一种有效的广谱抗生素，它通过抑制细菌蛋白质的合成而起作用，然而，过量使用和残留氯霉素会影响人体健康。一方面，过量的氯霉素会对造血系统和消化系统造成不可逆的损害；另一方面，食品中存在的药物残留也会严重威胁人类健康。因此，氯霉素已经在全球畜牧业中被禁止使用。目前，常用的氯霉素检测和分类方法包括酶联免疫吸附法(ELISA)、高效液相色谱法(HPLC)和免疫分析法。尽管上述方法检测精度高，但仍存在实验耗时长、设备昂贵、对操作者技能要求高、结果易受基质干扰产生假阴性和假阳性等问题，且不适用于现场即时检测。因此，设计一种高灵敏性的氯霉素的方法。
近年来，荧光方法因操作简单、精度高、可视化等优点被大量研究，这有利于突破环境限制，在不同条件下对氯霉素实时监测。因此，开发高灵敏性的氯霉素荧光检测探针具有重要的实际意义。镧系材料具有尖锐的线状发射带和长的荧光寿命，特别是基于铕离子(Eu3+)的配位聚合物具有良好的红色荧光发射，适用于荧光定量和可视化检测。但是，基于铕离子的单信号容易受到环境和仪器的干扰，这使得荧光探针的稳定性和重现性较差。因此，迫切需要设计和开发一种稳定性好、重现性好且能用于氯霉素检测的比率型荧光探针。
发明内容
针对现有技术中存在不足，本发明提供了一种复合磁助比率荧光探针及其制备方法和应用；本发明所述复合磁助比率荧光探针由荧光微球探针和磁功能化的铕聚合物复合得到，随着氯霉素的加入可观察到复合磁助比率荧光探针的荧光信号变化；所述复合磁助比率荧光探针具有内置校准功能，能降低环境和仪器带来的干扰，并且其对氯霉素有良好的选择性，对常见的抗生素、生理物质、无机离子具有良好的抗干扰性能，能够用于可视化检测食品中的氯霉素。
本发明首先提供了一种复合磁助比率荧光探针，所述复合磁助比率荧光探针由荧光微球探针和磁功能化的铕聚合物复合得到；所述荧光微球探针形状接近球体，表面粗糙；所述磁功能化的铕聚合物自组装制备得到，其形状接近球体，有明显的层状结构；所述荧光微球探针在磁功能化的铕聚合物的不同层面分布均匀。
本发明还提供了上述复合磁助比率荧光探针的制备方法，具体包括如下步骤：
(1)BCDs@CaCO3-Apt荧光微球探针的制备：
将柠檬酸和聚乙烯亚胺(PEI)超声混合后在恒温加热条件下进行反应1，反应1结束后冷却、洗脱、纯化，干燥得到BCDs；将CaCl2、羧甲基纤维素(CMC)和BCDs混合均匀，然后加入Na2CO3水溶液进行矿化反应2，矿化反应2结束后然后离心、洗涤、干燥得到BCDs@CaCO3；
将BCDs@CaCO3、N-羟基磺基琥珀酰亚胺(NHS)、碳二亚胺盐酸盐(EDC)、氯霉素适配体(CAP-Apt)分散在MES缓冲液中混匀进行反应3，反应3结束后离心、洗涤，最终得到所述的BCDs@CaCO3-Apt荧光探针。
其中，所述柠檬酸和PEI的质量比为2:1；所述BCDs反应1的条件为在200～250℃下反应6～10h；
所述CaCl2、CMC、BCDs和Na2CO3的质量比为1:1:1.5:1，矿化反应2的条件为室温下反应24～30h。
所述BCDs@CaCO3、NHS、EDC的质量比为5～15:2～8:3～12，反应3的反应时间为4～10h，混合溶液中CAP-Apt的终浓度为0.1～1mg/mL。
(2)磁功能化的铕配位聚合物的制备：
S1.Fe3O4@SiO2的制备：
将六水合氯化铁和柠檬酸钠溶于乙二醇中，向其中加入醋酸钠并在恒温加热条件下进行反应4，反应4结束后冷却、洗涤、干燥，得到Fe3O4；
向Fe3O4的乙醇水溶液中加入氨水和正硅酸四乙酯(TEOS)，然后进行搅拌反应5，反应5结束后冷却、洗涤得到Fe3O4@SiO2。
其中，所述六水合氯化铁、柠檬酸钠和醋酸钠的质量比为3:1:6，六水合氯化铁与乙二醇加入比为0.3g:10mL；反应4的条件为在200～250℃下反应10～16h；
Fe3O4与乙醇水溶液1:5混合，乙醇水溶液中乙醇的体积分数为80％，氨水和正硅酸四乙酯的体积比为1:2，搅拌反应5的时间为12～15h。
S2.铕配位聚合物的制备：
将苯甲酰三氟丙酮(TTA)和2，2'-联嘧啶(bpm)加入乙醇溶液中溶解后，缓慢加入六水合氯化铕乙醇溶液混合均匀得到混合反应体系，然后混合反应体系调节pH至6～7后进行陈化反应6，反应结束后抽滤、洗涤、干燥，得到所述铕配位聚合物，记为[Eu(TTA)3]2bpm；
其中，六水合氯化铕乙醇溶液、苯甲酰三氟丙酮(TTA)、2，2'-联嘧啶(bpm)质量比为2～8:5～10:1～4；
所述混合反应体系中的乙醇总体积与六水合氯化铕乙醇溶液、苯甲酰三氟丙酮和2，2'- 联嘧啶的体积和相同；
所述陈化反应6的反应时间为1～4h。
S3.磁功能化的铕配位聚合物的制备：
将Fe3O4@SiO2与N，N-二甲基甲酰胺(DMF)混合均匀并调节pH至中性，然后向其中加入[Eu(TTA)3]2bpm和氨水，超声混合均匀，然后加入TEOS进行搅拌反应7，反应结束后、离心、洗涤、干燥，得到Eu-Fe3O4@SiO2磁性荧光微球；
将3-氨丙基三乙氧基硅烷(APTES)溶液加入Eu-Fe3O4@SiO2溶液中进行反应8，反应结束后洗涤干燥，得到Eu-Fe3O4@SiO2-NH2；
将氯霉素半抗原(CAP-Hapten)、N-羟基磺基琥珀酰亚胺(NHS)和碳二亚胺盐酸盐(EDC)在甲醇溶液中混合，加入Eu-Fe3O4@SiO2-NH2的MES缓冲液搅拌反应9、分离、洗涤，最终获得磁功能化的铕配位聚合物，记为Eu-Fe3O4@SiO2-Hapten。
其中，Fe3O4@SiO2、N，N-二甲基甲酰胺(DMF)、[Eu(TTA)3]2bpm、氨水和TEOS的用量比为20～60mg：80～240mL：10～30mg：1.25～3.75mL：1～5mL；
所述反应7的条件为超声搅拌10～20min，室温下反应10～15h；
所述3-氨丙基三乙氧基硅烷(APTES)溶液的溶剂为无水乙醇，Eu-Fe3O4@SiO2溶液的溶剂为纯水，3-氨丙基三乙氧基硅烷(APTES)溶液与Eu-Fe3O4@SiO2溶液的体积比为10～50:1～5；
反应8的条件为室温反应10～15h；
所述氯霉素半抗原(CAP-Hapten)、N-羟基磺基琥珀酰亚胺(NHS)、碳二亚胺盐酸盐(EDC)和Eu-Fe3O4@SiO2的质量比为1～5:2～10:3～15:10～50；
所述反应9的条件为搅拌反应4～8h。
(3)复合磁助比率荧光探针的制备：
将BCDs@CaCO3-Apt溶液和Eu-Fe3O4@SiO2-Hapten溶液混合均匀后即可得到复合磁助比率荧光探针溶液，记为BCDs@CaCO3-Apt/Eu-Fe3O4@SiO2-Hapten。
其中，所述BCDs@CaCO3-Apt溶液和Eu-Fe3O4@SiO2-Hapten溶液的体积比为1～6:1～9；
所述BCDs@CaCO3-Apt溶液浓度为10mg/mL，Eu-Fe3O4@SiO2-Hapten溶液浓度为10mg/mL，即所述BCDs@CaCO3-Apt溶液的浓度和Eu-Fe3O4@SiO2-Hapten溶液的浓度相同。
本发明中还提供了上述复合磁助比率荧光探针在可视化检测氯霉素中的应用，所述可视化检测为半定量检测或定量检测。
进一步的，所述应用具体包括如下步骤：
(1)氯霉素检测标准曲线的绘制：
将氯霉素水溶液与BCDs@CaCO3-Apt/Eu-Fe3O4@SiO2-Hapten混合得到荧光复合体系，通过荧光分析法拟合出氯霉素浓度的标准曲线，并记录荧光复合体系在紫外灯下的颜色情况。
(2)将待测样品与比率荧光复合体系混合后放置在紫外灯下，记录颜色信息，得到氯霉素浓度范围；然后检测混合液的荧光强度值，根据标准曲线得出氯霉素浓度。
与现有技术相比，本发明的有益效果在于：
1.本发明的复合磁助比率荧光探针运用竞争法进行检测，通过免疫氯霉素半抗原与氯霉素竞争氯霉素适配体进行检测，氯霉素半抗原能与适配体结合，但结合能力弱于氯霉素；适配体具有较强的特异性，相较于抗体和酶，有高的稳定性和环境适应性。与现有技术相比，本发明所述方法的检测时间相比其他荧光分析法缩短了一半，检测限从现有检测方法的0.7ng/mL精确到0.09ng/mL。
2.本发明的复合磁助比率荧光探针可观察到两种荧光信号，随着氯霉素的加入，其中一种荧光信号会发生变化，由于免疫反应，BCDs@CaCO3-Apt优先与氯霉素结合，在磁场作用下，磁功能化的铕聚合物保留，蓝色荧光在冲洗后被去除，因此蓝色荧光减弱，红色荧光不变。相比单荧光信号探针，本发明的优势是能降低环境和仪器带来的干扰。
3.本发明的复合磁助比率荧光探针与不同浓度氯霉素混合后在紫外灯下可以产生梯度的颜色变化，随着氯霉素浓度的增加，混合体系的荧光颜色发生变化。根据颜色可半定量判断出氯霉素浓度范围，更易于实现待测物的可视化检测。
4.本发明的复合磁助比率荧光探针对氯霉素有良好的选择性，对常见的抗生素和无机离子具有良好的抗干扰性能，在食品检测方面具有一定的实用价值。
5.本发明所述磁功能化的铕配位聚合物在铕的二元配合物中引进另一个配位体——2，2'-联嘧啶(bpm)形成三元配合物，使原配合物的发光强度得到极大提高，铕离子发光性质在双配体的作用下得到增强和修饰，从而提高稀土配合物的发光效率。本发明中将碳点和铕聚合物结合形成的比率荧光带有自校准功能，能消除外界因素带来的波动，提高检测结果的可靠性和重现性。
附图说明
图1是本发明所得BCDs@CaCO3-Apt的透射电镜图(A)和激光共聚焦图(B)，图中1-8分别为不同层面的单球荧光共聚焦图片。
图2是本发明所得Eu-Fe3O4@SiO2-Hapten的透射电镜图(A)和激光共聚焦图(B)，图1-4分别为不同层面的单球荧光共聚焦图片。
图3是加入不同浓度氯霉素后复合磁助比率荧光探针的荧光图谱。
图4为荧光强度比值F435/F615与氯霉素浓度拟合的校准曲线。
图5是不同浓度氯霉素对应比率荧光探针的荧光颜色图。
图6是本发明所得比率荧光探针的选择性示意图；其中A为复合磁助比率荧光探针对不同种抗生素(头孢匹林、阿莫西林、四环素、恩氟沙星和磺胺甲恶唑)的选择性示意图，B为复合磁助比率荧光探针对不同种无机离子(钾离子、钠离子、镁离子、锌离子、碳酸根离子、硫酸根离子、硝酸根离子)的选择性示意图。
具体实施方式
下面结合附图以及具体实施例对本发明作进一步的说明，但本发明的保护范围并不限于此。以下实施例中所述试剂如无特殊说明，均可通过商业购买获得。
实施例1：复合磁助比率荧光探针的制备
(1)BCDs@CaCO3-Apt荧光微球探针的制备：
将1.0g的柠檬酸和0.5g的PEI溶解在30mL超纯水中超声混合，将混合物转移到50mL特氟龙内衬中，200℃加热6h后冷却至室温，产物用甲醇洗脱、用硅胶色谱柱纯化，在旋转蒸发仪中干燥获得浓缩BCDs溶液，通过冷冻浓缩溶液(-80℃)，然后真空干燥，得到BCDs。
将33.4mg CaCl2、30mg CMC和50mg BCDs溶于30mL水中恒定速率搅拌30min，然后加入31.8mg Na2CO3水矿化反应24h，溶液5000rpm离心10min，纯水洗涤后真空干燥，得到BCDs@CaCO3。
将50mg BCDs@CaCO3、10mg NHS、15mg EDC分散在5mL MES缓冲液(pH＝6.2)中反应4h，加入100μL的CAP-Apt(5.0mg/mL，购自上海生工生物试剂有限公司)，再反应4h后，以5000rpm离心10min，用纯水洗涤3次，最终获得BCDs@CaCO3-Apt荧光探针溶液。
如图1透射电镜图所示，制备的BCDs@CaCO3-Apt大小分布均匀，没有聚集，粒径为675～925nm，由激光共聚焦图可以看到荧光分布均匀。
(2)磁功能化的铕配位聚合物的制备：
S1.Fe3O4@SiO2的制备：
将0.65g FeCl3·6H2O和0.20g柠檬酸钠溶解在20mL乙二醇中，再加入1.20g NaAc搅拌30min，将混合液转入50mL特氟龙内衬中，200℃加热10h，冷却至室温后，产品用乙醇和去离子水分别洗涤3次，在60℃真空下干燥6h得到Fe3O4。
将20mg Fe3O4溶于80mL乙醇和20mL水中，加入1.25mL氨水，超声分散10min后加入2.5mL的TEOS，搅拌12h，产物用乙醇洗涤3次，获得Fe3O4@SiO2。
S2.铕配位聚合物的制备：
0.73g EuCl3·6H2O溶于20mL乙醇中；1.30g TTA，0.20g bpm溶于另外20mL乙醇， 置于50℃油浴中，同时磁力搅拌。然后将EuCl3·6H2O的乙醇溶液缓慢滴入配体溶液中，用NaOH调节pH＝6～7，继续反应1h，陈化、抽滤、洗涤、干燥，得到铕配位聚合物，记为[Eu(TTA)3]2bpm。
S3.磁功能化的铕配位聚合物的制备：
将20mg Fe3O4@SiO2加入80mL DMF和20mL纯水中，用0.1mol/L的HCl将溶液调到中性，然后加入10mg[Eu(TTA)3]2bpm和1.25mL氨水，超声10min后加入2.5mL TEOS，继续反应12h，用无水乙醇洗涤3次，干燥后得到Eu-Fe3O4@SiO2。将50mg Eu-Fe3O4@SiO2分散于50mL超纯水中，加入2mL APTES后在摇床上反应12h，用乙醇和超纯水分别洗涤3次后50℃真空干燥8h。将5.0mg CAP-Hapten(购自上海生工生物试剂有限公司)、10mg NHS和15mg EDC溶解在1mL甲醇中反应4h，将混合物加入含有50.0mg Eu-Fe3O4@SiO2的MES缓冲液(5mL)中搅拌3h，洗涤后获得磁功能化的铕配位聚合物，记为Eu-Fe3O4@SiO2-Hapten。
图2是Eu-Fe3O4@SiO2-Hapten的透射电镜图(A)和激光共聚焦图(B)；如透射电镜图所示，通过自组装制备的铕配位聚合物具有清晰层状结构，由激光共聚焦图可以看到各层都有较强的荧光。
(3)复合磁助比率荧光探针的制备：
将30μL的Eu-Fe3O4@SiO2-Hapten溶液和20μL的BCDs@CaCO3-Apt溶液混合，其中，所述BCDs@CaCO3-Apt溶液浓度为10mg/mL，Eu-Fe3O4@SiO2-Hapten溶液浓度为10mg/mL，得到复合磁助比率荧光探针，记为BCDs@CaCO3-Apt/Eu-Fe3O4@SiO2-Hapten。
实施例2：复合磁助比率荧光探针的制备
(1)BCDs@CaCO3-Apt荧光微球探针的制备：
将1.0g的柠檬酸和0.5g的PEI溶解在30mL超纯水中超声混合，将混合物转移到50mL特氟龙内衬中，220℃加热8h后冷却至室温，产物用甲醇洗脱、用硅胶色谱柱纯化，在旋转蒸发仪中干燥获得浓缩BCDs溶液，通过冷冻浓缩溶液(-80℃)，然后真空干燥，得到BCDs。
将33.4mg CaCl2、30mg CMC和50mg BCDs溶于30mL水中恒定速率搅拌30min，加入31.8mg Na2CO3水矿化反应24h，溶液5000rpm离心10min，纯水洗涤后真空干燥。
将10mg BCDs@CaCO3、10mg NHS、10mg EDC分散在5mL MES缓冲液(pH＝6.2)中反应10h，加入80μL的CAP-Apt(5.0mg/mL)，再反应4h后，以5000rpm离心10min，用纯水洗涤3次，最终获得BCDs@CaCO3-Apt荧光探针溶液。
(2)磁功能化的铕配位聚合物的制备：
S1.Fe3O4@SiO2的制备：
将0.65g FeCl3·6H2O和0.20g柠檬酸钠溶解在20mL乙二醇中，再加入1.20g NaAc搅拌30min，将混合液转入50mL特氟龙内衬中，200℃加热10h，冷却至室温后，产品用乙醇和去离子水分别洗涤3次，在60℃真空下干燥6h得到Fe3O4。
将20mg Fe3O4溶于80mL乙醇和20mL水中，加入1.25mL氨水，超声分散10min后加入2.5mL的TEOS，搅拌12h，产物用乙醇洗涤3次，获得Fe3O4@SiO2。
S2.铕配位聚合物的制备：
0.4g EuCl3·6H2O溶于20mL乙醇中；0.8g TTA，0.20g bpm溶于另外20mL乙醇，置于50℃油浴中，同时磁力搅拌。然后将EuCl3·6H2O的乙醇溶液缓慢滴入配体溶液中，用NaOH调节pH＝6～7，继续反应2h，陈化、抽滤、洗涤、干燥，得到铕配位聚合物，记为[Eu(TTA)3]2bpm。
S3.磁功能化的铕配位聚合物的制备：
将20mg Fe3O4@SiO2加入50mL DMF和20mL纯水中，用0.1mol/L的HCl将溶液调到中性后加入15mg[Eu(TTA)3]2bpm和2mL氨水，超声10min，加入3mL TEOS，继续反应15h，用无水乙醇洗涤3次，干燥后得到Eu-Fe3O4@SiO2。将50mg Eu-Fe3O4@SiO2分散于50mL超纯水中，加入5mL APTES后在摇床上反应15h，用乙醇和超纯水分别洗涤3次后50℃真空干燥8h。将5.0mg CAP-Hapten、15mg NHS和20mg EDC溶解在1mL甲醇中反应4h，将混合物加入含有80.0mg Eu-Fe3O4@SiO2的MES缓冲液(5mL)中搅拌5h，洗涤后获得磁功能化的铕配位聚合物，记为Eu-Fe3O4@SiO2-Hapten。
(3)复合磁助比率荧光探针BCDs@CaCO3-Apt/Eu-Fe3O4@SiO2-Hapten的制备：
将10μL的Eu-Fe3O4@SiO2-Hapten溶液和30μL的BCDs@CaCO3-Apt溶液混合，其中，所述BCDs@CaCO3-Apt溶液浓度为10mg/mL，Eu-Fe3O4@SiO2-Hapten溶液浓度为10mg/mL，得到复合磁助比率荧光探针，记为BCDs@CaCO3-Apt/Eu-Fe3O4@SiO2-Hapten。
实施例3：复合磁助比率荧光探针的制备
(1)BCDs@CaCO3-Apt荧光微球探针的制备：
将1.0g的柠檬酸和0.5g的PEI溶解在30mL超纯水中超声混合，将混合物转移到50mL特氟龙内衬中，200℃加热6h后冷却至室温，产物用甲醇洗脱、用硅胶色谱柱纯化，在旋转蒸发仪中干燥获得浓缩BCDs溶液，通过冷冻浓缩溶液(-80℃)，然后真空干燥，得到BCDs。
将33.4mg CaCl2、30mg CMC和50mg BCDs溶于30mL水中恒定速率搅拌30min，加入31.8mg Na2CO3水矿化反应24h，溶液5000rpm离心10min，纯水洗涤后真空干燥。
将60mg BCDs@CaCO3、28mg NHS、40mg EDC分散在5mL MES缓冲液(pH＝6.2)中反应7h，加入120μL的CAP-Apt(5.0mg/mL)，再反应4h后，以5000rpm离心10min， 用纯水洗涤3次，最终获得BCDs@CaCO3-Apt荧光探针溶液。
(2)磁功能化的铕配位聚合物的制备：
S1.Fe3O4@SiO2的制备：
将0.65g FeCl3·6H2O和0.20g柠檬酸钠溶解在20mL乙二醇中，再加入1.20g NaAc搅拌30min，将混合液转入50mL特氟龙内衬中，220℃加热12h，冷却至室温后，产品用乙醇和去离子水分别洗涤3次，在60℃真空下干燥6h得到Fe3O4。
将20mg Fe3O4溶于80mL乙醇和20mL水中，加入1.25mL氨水，超声分散10min后加入2.5mL的TEOS，搅拌12h，产物用乙醇洗涤3次，获得Fe3O4@SiO2。
S2.铕配位聚合物的制备：
1.4g EuCl3·6H2O溶于20mL乙醇中；1.8g TTA，0.6g bpm溶于另外20mL乙醇，置于50℃油浴中，同时磁力搅拌。然后将EuCl3·6H2O的乙醇溶液缓慢滴入配体溶液中，用NaOH调节pH＝6～7，继续反应4h，陈化、抽滤、洗涤、干燥，得到铕配位聚合物，记为[Eu(TTA)3]2bpm。
S3.磁功能化的铕配位聚合物的制备：
将20mg Fe3O4@SiO2加入130mL DMF和20mL纯水中，用0.1mol/L的HCl将溶液调到中性后加入30mg[Eu(TTA)3]2bpm和3mL氨水，超声10min，加入5mL TEOS，继续反应10h，用无水乙醇洗涤3次，干燥后得到Eu-Fe3O4@SiO2。将80mg Eu-Fe3O4@SiO2分散于50mL超纯水中，加入8mL APTES后在摇床上反应10h，用乙醇和超纯水分别洗涤3次后50℃真空干燥8h。将8.0mg CAP-Hapten、40mg NHS和80mg EDC溶解在1mL甲醇中反应4h，将混合物加入含有100.0mg Eu-Fe3O4@SiO2的MES缓冲液(5mL)中搅拌8h，洗涤后获得磁功能化的铕配位聚合物，记为Eu-Fe3O4@SiO2-Hapten。
(3)复合磁助比率荧光探针BCDs@CaCO3-Apt/Eu-Fe3O4@SiO2-Hapten的制备：
将10μL的Eu-Fe3O4@SiO2-Hapten溶液和25μL的BCDs@CaCO3-Apt溶液混合，其中，所述BCDs@CaCO3-Apt溶液浓度为10mg/mL，Eu-Fe3O4@SiO2-Hapten溶液浓度为10mg/mL，得到复合磁助比率荧光探针，记为BCDs@CaCO3-Apt/Eu-Fe3O4@SiO2-Hapten。
实施例4：氯霉素检测标准曲线绘制
用PBS配制氯霉素标准溶液，将100μL氯霉素水溶液与实施例1所述的复合磁助比率荧光探针BCDs@CaCO3-Apt/Eu-Fe3O4@SiO2-Hapten混合后得到氯霉素浓度分别为0，0.1，0.5，1，3，5，7，10，15和20ng/mL的复合体系，在常温状态下孵育10min，除去混合物的上清液，沉淀物由磁铁收集，随后再分散在1mL的PBS中，在365nm激发光下，记录不同氯霉素浓度下的复合体系在350～650nm下的荧光光谱。
如图3所示，当没有氯霉素存在时，复合磁助比率荧光探针同时发出红色荧光和蓝色荧光，随着氯霉素的加入可观察到荧光信号变化，由于免疫反应，BCDs@CaCO3-Apt优先与氯霉素结合，在磁场作用下，磁功能化的红色荧光保留，蓝色荧光在冲洗后被去除，因此蓝色荧光减弱。
图4是根据435nm和615nm处荧光强度比值F435/F615与氯霉素浓度变化拟合的校准曲线，对应的函数为Y＝6.663-0.304X，相关系数R2＝0.997，线性范围为0～20ng/mL。
实施例5：氯霉素半定量可视化检测
将实施例1中获得的复合磁助比率荧光探针在不同氯霉素浓度下比率荧光复合，孵育10min后除去上清液，由磁铁收集沉淀物，随后将沉淀物分散在PBS中，放置在紫外灯下，记录上述溶液对应的颜色信息，将颜色信号与氯霉素浓度分别对应，按照从小到大的顺序记录颜色变化。
图5为荧光强度比值F435/F615与氯霉素浓度拟合的校准曲线，从图中可以看出，在365nm紫外光下，通过肉眼可以清晰地看到，添加不同浓度氯霉素浓度(0～20ng/mL)后，混合体系的荧光颜色从紫色变到红色。因此，本发明所述的复合磁助比率荧光探针根据颜色信息可实现氯霉素的半定量可视化检测。
实施例6：牛奶样品中氯霉素的检测分析
为了测试智能手机与复合磁助比率荧光探针结合后在实际样品中的定量分析效果，本实施例选择待测样本检测残留情况，具体步骤如下所示：
在2mL样品中加入20μL的三氯乙酸(20％V/V)，超声处理30min，并在10000rpm下离心5min，用滤纸过滤上清液，得到含有氯霉素的提取液；将提取液加入到实施例1所述的复合比率荧光探针中，孵育10min后除去混合物的上清液，沉淀物是由磁铁收集，随后再分散在PBS中，溶液放置在365nm紫外灯下，观察到溶液颜色。将上述溶液放置在荧光光谱仪中，在360nm激发光下测定荧光光谱，得到荧光强度比值F435/F615，将得到的F435/F615带入到实施例4所述的拟合方程中得到样品中氯霉素含量。用标准加标方法探索复合磁助比率荧光探针在氯霉素检测中的准确性，结果显示加标氯霉素可以准确测量，回收率较高(从97％到107％)，精密度较好(RSD<5.2％)。
实施例7：复合磁助比率荧光探针的特异性评估
本实施例中分别配制了种抗生素(头孢匹林、阿莫西林、四环素、恩氟沙星和磺胺甲恶唑)、无机离子(钾离子、钠离子、镁离子、锌离子、碳酸根离子、硫酸根离子、硝酸根离子)等标准溶液作为干扰物质测试评估复合磁助比率荧光探针的特异性，浓度均为1M。
将不同种抗生素分别加入到实施例1所述的复合比率荧光探针溶液中，最终抗生素的浓 度均为10ng/mL，采用实施例5所述方法进行检测，检测结果如图6A所示。
图6A是本发明所得复合磁助比率荧光探针对头孢匹林、阿莫西林、四环素、恩氟沙星和磺胺甲恶唑这五种抗生素的选择性示意图，从图中可以看出，荧光比值变化仅在含有CAP而不是其他加标样品的溶液中发现，说明适配体识别具有优异的选择性，也说明该复合体系检测氯霉素具有特异性，不会被共存的其他抗生素干扰。
将氯霉素分别与不同种无机离子混合，将混合液分别加入到实施例1所述的复合比率荧光探针溶液中得到复合体系，最终复合体系中无机离子的浓度均为500μM，氯霉素的浓度为50μM，采用实施例5所述方法进行检测，检测结果如图6B所示。
图6B是复合比率荧光探针对钾离子、钠离子、镁离子、锌离子、碳酸根离子、硫酸根离子、硝酸根离子的选择性示意图，从图中可以看出，混合液的信号与氯霉素单独存在时的信号未观察到显著变化，说明该传感器对氯霉素具有良好的选择性，不会受到溶液中其他离子的干扰。
综上，成功构建了一种复合磁助比率荧光探针，实现了氯霉素的高灵敏度和高选择性检测。
所述实施例为本发明的优选的实施方式，但本发明并不限于上述实施方式，在不背离本发明的实质内容的情况下，本领域技术人员能够做出的任何显而易见的改进、替换或变型均属于本发明的保护范围。

Claims (10)

  1. 一种复合磁助比率荧光探针，其特征在于，所述复合磁助比率荧光探针由荧光微球探针和磁功能化的铕聚合物复合得到；所述荧光微球探针形状接近球体，表面粗糙；所述磁功能化的铕聚合物自组装制备得到，其形状接近球体，有明显的层状结构；所述荧光微球探针在磁功能化的铕聚合物的不同层面分布均匀。
  2. 权利要求1所述的复合磁助比率荧光探针的制备方法，其特征在于，
    (1)荧光微球探针的制备：
    将BCDs@CaCO3、N-羟基磺基琥珀酰亚胺、碳二亚胺盐酸盐、氯霉素适配体分散在MES缓冲液中混匀得到混合液，然后将混合液室温下反应，反应结束后离心、洗涤，得到所述荧光微球探针，记为BCDs@CaCO3-Apt；
    (2)磁功能化的铕配位聚合物的制备：
    S1.铕配位聚合物的制备：
    将苯甲酰三氟丙酮和2，2'-联嘧啶加入乙醇溶液中溶解后，缓慢加入六水合氯化铕乙醇溶液混合均匀得到混合反应体系，然后混合反应体系调节pH至6～7后进行陈化反应，反应结束后抽滤、洗涤、干燥，得到所述铕配位聚合物，记为[Eu(TTA)3]2bpm；
    S2.磁功能化的铕配位聚合物的制备：
    将Fe3O4@SiO2与N，N-二甲基甲酰胺混合均匀并调节pH至中性，然后向其中加入[Eu(TTA)3]2bpm和氨水，超声混合均匀，然后加入TEOS进行第一搅拌反应，反应结束后、离心、洗涤、干燥，得到Eu-Fe3O4@SiO2；
    将3-氨丙基三乙氧基硅烷溶液加入Eu-Fe3O4@SiO2溶液中进行反应，反应结束后洗涤干燥，得到Eu-Fe3O4@SiO2-NH2；
    将氯霉素半抗原、N-羟基磺基琥珀酰亚胺和碳二亚胺盐酸盐在甲醇溶液中混合，加入Eu-Fe3O4@SiO2-NH2的MES缓冲液进行第二搅拌反应，反应结束后分离、洗涤，最终获得磁功能化的铕配位聚合物，记为Eu-Fe3O4@SiO2-Hapten；
    (3)复合磁助比率荧光探针的制备：
    将BCDs@CaCO3-Apt溶液和Eu-Fe3O4@SiO2-Hapten溶液混合均匀后即可得到复合磁助比率荧光探针溶液，记为BCDs@CaCO3-Apt/Eu-Fe3O4@SiO2-Hapten。
  3. 根据权利要求2所述的复合磁助比率荧光探针的制备方法，其特征在于，步骤(1)中，所述BCDs@CaCO3、N-羟基磺基琥珀酰亚胺和碳二亚胺盐酸盐的质量比为5～15:2～8:3～12；
    所述室温下反应的时间为4～10h；
    所述混合液中的氯霉素适配体的终浓度为0.1～1mg/mL。
  4. 根据权利要求2所述的复合磁助比率荧光探针的制备方法，其特征在于，步骤S1中，六水合氯化铕乙醇溶液、苯甲酰三氟丙酮和2，2'-联嘧啶质量比为2～8:5～10:1～4；
    所述混合反应体系中的乙醇总体积与六水合氯化铕乙醇溶液、苯甲酰三氟丙酮和2，2'-联嘧啶的体积和相同；
    所述陈化反应的反应时间为1～4h。
  5. 根据权利要求2所述的复合磁助比率荧光探针的制备方法，其特征在于，步骤S2中Eu-Fe3O4@SiO2制备时：
    所述Fe3O4@SiO2、N，N-二甲基甲酰胺、[Eu(TTA)3]2bpm、氨水和TEOS的用量比为20～60mg：80～240mL：10～30mg：1.25～3.75mL：1～5mL；
    所述第一搅拌反应的条件为：超声搅拌10～20min后，在室温下反应10～15h。
  6. 根据权利要求2所述的复合磁助比率荧光探针的制备方法，其特征在于，步骤S2中Eu-Fe3O4@SiO2-NH2制备时：
    所述3-氨丙基三乙氧基硅烷溶液与Eu-Fe3O4@SiO2溶液的体积比为10～50:1～5；所述3-氨丙基三乙氧基硅烷溶液的溶剂为无水乙醇，Eu-Fe3O4@SiO2溶液的溶剂为纯水，
    所述反应条件为室温下反应10～15h。
  7. 根据权利要求2所述的复合磁助比率荧光探针的制备方法，其特征在于，步骤S2中Eu-Fe3O4@SiO2-Hapten制备时：
    所述氯霉素半抗原CAP-Hapten、N-羟基磺基琥珀酰亚胺、碳二亚胺盐酸盐和Eu-Fe3O4@SiO2的质量比为1～5:2～10:3～15:10～50；
    所述第二搅拌反应的条件为搅拌反应4～8h。
  8. 根据权利要求2所述的复合磁助比率荧光探针的制备方法，其特征在于，步骤(3)中，所述BCDs@CaCO3-Apt溶液和Eu-Fe3O4@SiO2-Hapten溶液的体积比为1～6:1～9；
    所述BCDs@CaCO3-Apt溶液的浓度和Eu-Fe3O4@SiO2-Hapten溶液的浓度相同。
  9. 权利要求2～8所述方法制备的复合磁助比率荧光探针或权利要求1所述的复合磁助比率荧光探针在可视化检测氯霉素中的应用。
  10. 权利要求9所述的应用，其特征在于，所述可视化检测为半定量检测或定量检测。
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101225305A (zh) * 2007-12-29 2008-07-23 中国科学院长春应用化学研究所 一种兼具磁性和稀土特征发光性能的纳米材料及制备方法
US8722415B1 (en) * 2010-10-07 2014-05-13 U.S. Department Of Energy Method of detecting luminescent target ions with modified magnetic microspheres
CN108535486A (zh) * 2018-02-11 2018-09-14 北京华海骏业科技发展有限公司 一种基于铕标记的氯霉素免疫荧光测定方法
CN110776647A (zh) * 2019-08-28 2020-02-11 浙江工业大学 一种基于铕金属有机框架的比率探针及其构建方法和应用
CN112798565A (zh) * 2020-12-30 2021-05-14 张磊 一种磁功能化比率型稀土荧光探针及制备与应用
CN113788984A (zh) * 2021-08-04 2021-12-14 广东碳紫科技有限公司 一种碳量子点-碳酸钙复合纳米颗粒及其制备方法与应用
CN114149592A (zh) * 2021-10-20 2022-03-08 江苏大学 一种复合比率荧光探针及其制备方法和应用
CN116120920A (zh) * 2023-02-16 2023-05-16 江苏大学 一种复合磁助比率荧光探针及其制备方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103550834B (zh) * 2013-10-25 2015-07-15 北京大学 一种栓塞材料组合物及其制备方法和用途

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101225305A (zh) * 2007-12-29 2008-07-23 中国科学院长春应用化学研究所 一种兼具磁性和稀土特征发光性能的纳米材料及制备方法
US8722415B1 (en) * 2010-10-07 2014-05-13 U.S. Department Of Energy Method of detecting luminescent target ions with modified magnetic microspheres
CN108535486A (zh) * 2018-02-11 2018-09-14 北京华海骏业科技发展有限公司 一种基于铕标记的氯霉素免疫荧光测定方法
CN110776647A (zh) * 2019-08-28 2020-02-11 浙江工业大学 一种基于铕金属有机框架的比率探针及其构建方法和应用
CN112798565A (zh) * 2020-12-30 2021-05-14 张磊 一种磁功能化比率型稀土荧光探针及制备与应用
CN113788984A (zh) * 2021-08-04 2021-12-14 广东碳紫科技有限公司 一种碳量子点-碳酸钙复合纳米颗粒及其制备方法与应用
CN114149592A (zh) * 2021-10-20 2022-03-08 江苏大学 一种复合比率荧光探针及其制备方法和应用
CN116120920A (zh) * 2023-02-16 2023-05-16 江苏大学 一种复合磁助比率荧光探针及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ILMI RASHID, SUN WEIDONG, DUTRA JOSÉ D. L., AL-RASBI NAWAL K., ZHOU LIANG, QIAN PENG-CHENG, WONG WAI-YEUNG, RAITHBY PAUL R., KHAN : "Monochromatic red electroluminescence from a homodinuclear europium(III) complex of a β-diketone tethered by 2,2'-bipyrimidine", JOURNAL OF MATERIALS CHEMISTRY C, ROYAL SOCIETY OF CHEMISTRY, GB, vol. 8, no. 29, 30 July 2020 (2020-07-30), GB , pages 9816 - 9827, XP093201980, ISSN: 2050-7526, DOI: 10.1039/D0TC02181D *

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