Composite magnetic assisted ratiometric fluorescent probe and preparation method and use thereof

Abstract

The present invention belongs to the technical field of preparation of nano materials and sensors. Provided are a composite magnetic-assisted ratiometric fluorescent probe, a preparation method theref

Description

The present disclosure belongs to the technical field of nanomaterial and sensor preparation, and specifically relates to a composite magnetic-assisted ratiometric fluorescent probe and a preparation method and use thereof. BACKGROUND Chloramphenicol (CAP) is an effective broad-spectrum antibiotic. CAP works by inhibiting the synthesis of bacterial proteins. However, the excessive use of CAP and the residual CAP can affect the human health. On the one hand, the excessive CAP can cause an irreversible damage to the hematopoietic and digestive systems. On the other hand, the presence of a drug residue in the food can pose a serious threat to the human health. Therefore, CAP has been banned in the animal husbandry worldwide. Currently, the common methods for detecting and classifying CAP include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and immunoassay. These methods have high detection accuracy, but are still faced with problems such as long experimental time, expensive equipment, high requirements for operator skills, and false-negative and false-positive results easily caused by matrix interference. In addition, these methods are not suitable for on-site point-of-care testing. Therefore, a high-sensitivity method for detecting CAP needs to be designed. In recent years, fluorescence methods have been widely studied due to advantages such as simple operations, high accuracy, and visualization, which are conducive to the breakthrough of environmental limitations and the real-time monitoring of CAP under different conditions. Therefore, the development of high-sensitivity fluorescent probes for detecting CAP is of great practical significance. Lanthanide materials have sharp linear emission bands and long fluorescence lifetimes. In particular, europium ion (Eu3+)-based coordination polymers have prominent red fluorescence emissions, and are suitable for fluorescence quantification and visual detection. However, europium ion-based single signals are easily interfered with by environments and instruments, which results in poor stability and reproducibility of fluorescent probes. Therefore, there is an urgent need to design and develop a ratiometric fluorescent probe that has prominent stability and reproducibility and can be used for CAP detection. SUMMARY In view of the deficiencies in the prior art, the present disclosure provides a composite magnetic-assisted ratiometric fluorescent probe and a preparation method and use thereof. The composite magnetic-assisted ratiometric fluorescent probe of the present disclosure is produced by compounding fluorescent microsphere probes with a magnetically-functionalized europium coordination polymer. A fluorescence signal change of the composite magnetic-assisted ratiometric fluorescent probe can be observed with the addition of CAP. The composite magnetic-assisted ratiometric fluorescent probe has a built-in calibration function, can reduce the interference brought by an environment and an instrument, and exhibits excellent selectivity for CAP. The composite magnetic-assisted ratiometric fluorescent probe has a prominent anti-interference ability against common antibiotics, physiological substances, and inorganic ions, and can be used for visual detection of CAP in a food. The present disclosure provides a preparation method of a composite magnetic-assisted ratiometric fluorescent probe, where the composite magnetic-assisted ratiometric fluorescent probe is produced by compounding fluorescent microsphere probes with a magnetically-functionalized europium coordination polymer; the fluorescent microsphere probes each have a shape close to a sphere and a rough surface; the magnetically-functionalized europium coordination polymer is prepared through self-assembly, and has a shape close to a sphere and an obvious layered structure; and the fluorescent microsphere probes are uniformly distributed in different layers of the magnetically-functionalized europium coordination polymer, and where the preparation method of the composite magnetic-assisted ratiometric fluorescent probe specifically includes the following steps: (1) preparation of the fluorescent microsphere probes BCDs@CaCO3-Apt: ultrasonically mixing citric acid and polyethyleneimine (PEI), and heating to allow a first reaction at a constant temperature; after the first reaction, conducting cooling, elution, purification, and drying to obtain BCDs); thoroughly mixing CaCh, carboxymethylcellulose (CMC), and the BCDs, adding a Na2COs aqueous solution, and conducting a second mineralization reaction; and after the second mineralization reaction, conducting centrifugation, washing, and drying to obtain BCDs@CaCO3; evenly dispersing the BCDs@CaCO3, N-hydroxysulfosuccinimide (sulfo-NHS), l-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and a chloramphenicol aptamer (CAP-Apt) in a 2-morpholinoethanesulphonic acid (MES) buffer, thoroughly mixing to obtain a mixed solution, and subjecting the mixed solution to a third reaction; and after the third reaction, conducting centrifugation and washing to finally obtain the fluorescent microsphere probes BCDs@CaCO3-Apt, where a mass ratio of the citric acid to the PEI is 2:1, and the first reaction is conducted at 200°C to 250°C for 6 h to 10 h; the CaCh, the CMC, the BCDs, and Na2CO3 are in a mass ratio of 1:1:1.5:1, and the second mineralization reaction is conducted at room temperature for 24 h to 30 h; and the BCDs@CaCO3, the sulfo-NHS, and the EDC are in a mass ratio of (5 to 15):(2 to 8):(3 to 12), the third reaction is conducted for 4 h to 10 h, and a final concentration of the CAP-Apt in the mixed solution is 0.1 mg / mLto 1 mg / mL; (2) preparation of the magnetically-functionalized europium coordination polymer: SI, preparation of Fe3O4@SiO2: dissolving iron chloride hexahydrate and sodium citrate in ethylene glycol, adding sodium acetate, and heating to allow a fourth reaction at a constant temperature; and after the fourth reaction, conducting cooling, washing, and drying to obtain Fe3O4; adding ammonia water and tetraethyl orthosilicate (TEOS) to an aqueous ethanol solution of the Fe3O4, and conducting a fifth stirring-based reaction; and after the fifth stirring-based reaction, conducting cooling and washing to obtain the Fe3O4@SiO2, where the iron chloride hexahydrate, the sodium citrate, and the sodium acetate are in a mass ratio of 3:1:6, and an amount ratio of the iron chloride hexahydrate to the ethylene glycol is 0.3 g : 10 mL; the fourth reaction is conducted at 200°C to 250°C for 10 h to 16 h; the Fe3O4 is mixed with an aqueous ethanol solution in a ratio of 1:5, a volume fraction of ethanol in the aqueous ethanol solution is 80%, and a volume ratio of the ammonia water to the TEOS is 1:2; and the fifth stirring-based reaction is conducted for 12 h to 15 h; S2, preparation of a europium coordination polymer: dissolving benzoyl(trifluoroacetyl)methane (BTA) and 2,2'-bipyrimidine (bpm) in an ethanol solution, slowly adding an ethanol solution of europium chloride hexahydrate, and thoroughly mixing to obtain a mixed reaction system; adjusting a pH of the mixed reaction system to 6 to 7, and conducting a sixth aging reaction; and after the sixth aging reaction, conducting suction filtration, washing, and drying to obtain the europium coordination polymer, denoted as [Eu(BTA)3]2bpm, where the ethanol solution of the europium chloride hexahydrate, the BTA, and the bpm are in a mass ratio of (2 to 8):(5 to 10):(1 to 4); in the mixed reaction system, a total volume of ethanol is the same as a volume sum of the ethanol solution of the europium chloride hexahydrate, the BTA, and the bpm; and the sixth aging reaction is conducted for 1 h to 4 h; S3, preparation of the magnetically-functionalized europium coordination polymer: thoroughly mixing the Fe3O4@SiO2 with N,N-dimethylformamide (DMF), and adjusting a pH to 7.0; adding the [Eu(BTA)3]2bpm and ammonia water, and conducting ultrasonic mixing fully; adding TEOS, and conducting a seventh stirring-based reaction; after the seventh stirring-based reaction, conducting centrifugation, washing, and drying to obtain Eu-Fe3O4@SiO2; adding a 3-aminopropyltri ethoxy silane (APTES) solution to a Eu-Fe3O4@SiO2 solution to allow an eighth reaction, and after the eighth reaction, conducting washing and drying to obtain Eu-Fe3O4@SiO2-NH2; mixing a chloramphenicol hapten (CAP-Hapten), sulfo-NHS, and EDC in a methanol solution, adding a 2-morpholinoethanesulphonic acid (MES) buffer of the Eu-Fe3O4@SiO2-NH2, and conducting a ninth stirring-based reaction; and after the ninth stirring-based reaction, conducting separation and washing to finally obtain the magnetically-functionalized europium coordination polymer, denoted as Eu-Fe3O4@SiO2-Hapten, where the Fe3O4@SiO2, the DMF, the [Eu(BTA)3]2bpm, the ammonia water, and the TEOS are in an amount ratio of (20 to 60) mg : (80 to 240) mL : (10 to 30) mg : (1.25 to 3.75) mL : (1 to 5) mL; the seventh stirring-based reaction is conducted as follows: ultrasonically stirring for 10 min to 20 min, and allowing a reaction at room temperature for 10 h to 15 h; a solvent for the APTES solution is absolute ethanol and a solvent for the Eu-Fe3O4@SiO2 solution is pure water; a volume ratio of the APTES solution to the Eu-Fe3O4@SiO2 solution is (10 to 50):(1 to 5); the eighth reaction is conducted at room temperature for 10 h to 15 h; the CAP-Hapten, the sulfo-NHS, the EDC, and the Eu-Fe3O4@SiO2 are in a mass ratio of (1 to 5):(2 to 10):(3 to 15):(10 to 50); and the ninth stirring-based reaction is conducted under stirring for 4 h to 8 h; and (3) preparation of the composite magnetic-assisted ratiometric fluorescent probes: thoroughly mixing a BCDs@CaCO3-Apt solution with a Eu-Fe3O4@SiO2-Hapten solution to obtain a composite magnetic-assisted ratiometric fluorescent probe solution, denoted as B CD s@C aCOs - AptZEu-F e3 O4@ Si Ch-Hapten, where a volume ratio of the BCDs@CaCO3-Apt solution to the Eu-Fe3O4@SiO2-Hapten solution is (1 to 6):(1 to 9); and a concentration of the BCDs@CaCO3-Apt solution is 10 mg / mL, and a concentration of the Eu-Fe3O4@SiO2-Hapten solution is 10 mg / mL, namely, the BCDs@CaCO3-Apt solution and the Eu-Fe3O4@SiO2-Hapten solution have a same concentration. The present disclosure also provides a use of the composite magnetic-assisted ratiometric fluorescent probe in visual detection of CAP. The visual detection refers to semi-quantitative detection or quantitative detection. Further, the use specifically includes the following steps: (1) plotting of a standard curve for CAP detection: mixing CAP aqueous solutions with BCDs@CaCO3-Apt / Eu-Fe3O4@SiO2-Hapten to obtain fluorescence composite systems, fitting a standard curve of CAP concentrations through fluorescence analysis, and recording colors of the fluorescence composite systems under an ultraviolet lamp; and (2) mixing a sample to be tested with a ratiometric fluorescence composite system to obtain a mixed solution, placing the mixed solution under an ultraviolet lamp, and recording color information to obtain a CAP concentration range; and detecting a fluorescence intensity value of the mixed solution, and calculating a CAP concentration according to the standard curve. Compared with the prior art, the present disclosure has the following advantages: 1. The composite magnetic-assisted ratiometric fluorescent probe of the present disclosure allows a detection based on a competition law, where the CAP-Hapten competes with CAP for the CAP-Apt to implement detection. The CAP-Hapten can bind to the CAP-Apt, but exhibits a weaker binding ability than CAP. The CAP-Apt has strong specificity, and exhibits higher stability and environmental adaptability than antibodies and enzymes. A detection time of the method in the present disclosure is shortened by half compared with other fluorescence analysis methods, and a detection limit of the method in the present disclosure is accurate to 0.09 ng / mL from the 0.7 ng / mL of the existing detection method. 2. When the composite magnetic-assisted ratiometric fluorescent probe of the present disclosure is used, two fluorescence signals can be observed. With the addition of CAP, one of the two fluorescence signals will change. Due to an immune response, BCDs@CaCO3-Apt preferentially binds to CAP. Under an action of a magnetic field, the magnetically-functionalized europium coordination polymer is retained. Blue fluorescence is removed after rinsing. Therefore, the blue fluorescence is weakened, and the red fluorescence remains unchanged. Compared with a single-fluorescence-signal probe, the present disclosure has the advantage of reducing the interference caused by an environment and an instrument. 3. After being mixed with CAP of different concentrations, the composite magnetic-assisted ratiometric fluorescent probe of the present disclosure can produce a gradient color change under an ultraviolet lamp. A fluorescence color of a mixed system changes with the increase of a CAP concentration. Thus, a CAP concentration range can be determined semi-quantitatively according to a color, which makes it easy to achieve the visual detection of a sample to be tested. 4. The composite magnetic-assisted ratiometric fluorescent probe of the present disclosure exhibits prominent selectivity for CAP and a prominent anti-interference ability for common antibiotics and inorganic ions, and has a specified practical value in food detection. 5. In the magnetically-functionalized europium coordination polymer of the present disclosure, a ligand bpm is introduced into a europium-based binary complex to produce a ternary complex, which greatly improves a luminescence intensity of the original complex and enhances and modifies the luminescence properties of europium ions under an action of the double ligands, thereby improving a luminescence efficiency of the rare earth complex. The ratiometric fluorescence produced by combining carbon dots and a europium-based polymer in the present disclosure has a self-calibration function, which can eliminate the fluctuations caused by external factors and improve the reliability and reproducibility of detection results. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a transmission electron microscopy image (A) and confocal laser scanning microscopy images (B) of the BCDs@CaCO3-Apt obtained in the present disclosure, where 1 to 8 show confocal laser scanning microscopy images of single-sphere fluorescences at different levels, respectively; FIG. 2 shows a transmission electron microscopy image (A) and confocal laser scanning microscopy images (B) of the Eu-Fe3O4@SiO2-Hapten obtained in the present disclosure, where 1 to 4 show confocal laser scanning microscopy images of single-sphere fluorescences at different levels, respectively; FIG. 3 shows fluorescence spectra of the composite magnetic-assisted ratiometric fluorescent probe after CAP is added at different concentrations; FIG. 4 shows a calibration curve produced by fitting fluorescence intensity ratios F435 / F615 to CAP concentrations; FIG. 5 shows a fluorescence color map of the composite magnetic-assisted ratiometric fluorescent probe corresponding to CAP at different concentrations; and FIG. 6 shows schematic diagrams of selectivity of the composite magnetic-assisted ratiometric fluorescent probe obtained in the present disclosure, where A is a schematic diagram of selectivity of the composite magnetic-assisted ratiometric fluorescent probe for different antibiotics (cefapirin, amoxicillin, tetracycline, enrofloxacin, and sulfamethoxazole) and B is a schematic diagram of selectivity of the composite magnetic-assisted ratiometric fluorescent probe for different inorganic ions (potassium ions, sodium ions, magnesium ions, zinc ions, carbonate ions, sulfate ions, and nitrate ions). DETAILED DESCRIPTION OF THE EMBODIMENTS The present disclosure will be further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present disclosure is not limited thereto. Unless otherwise specified, the reagents in the following examples can be commercially available. Example 1 Preparation of a composite magnetic-assisted ratiometric fluorescent probe (1) Preparation of fluorescent microsphere probes BCDs@CaCO3-Apt 1.0 g of citric acid and 0.5 g of PEI were dissolved in 30 mL of ultrapure water, and ultrasonic mixing was conducted to obtain a mixture. The mixture was transferred into a 50 mL Teflon liner, heated at 200°C for 6 h, and cooled to room temperature to obtain a first product. The first product was subjected to elution with methanol, purification with a silica gel chromatographic column, and drying in a rotary evaporator to obtain a concentrated BCD solution. The concentrated BCD solution was frozen (-80°C) and then vacuum-dried to obtain BCDs. 33.4 mg of CaCh, 30 mg of CMC, and 50 mg of the BCDs were dissolved in 30 mL of water, and stirring was conducted at a constant rate for 30 min. Then 31.8 mg of Na2COs was added, and a mineralization reaction was conducted for 24 h to obtain a first reaction solution. The first reaction solution was centrifuged at 5,000 rpm for 10 min to obtain a first precipitate, and the first precipitate was washed with pure water and then vacuum-dried to obtain BCDs@CaCO3. 50 mg of the BCDs@CaCO3, 10 mg of sulfo-NHS, and 15 mg of EDC were dispersed in 5 mL of an MES buffer (pH = 6.2), and a reaction was conducted for 4 h. 100 pL of CAP-Apt (5.0 mg / mL, purchased from Sangon Biotech (Shanghai) Co., Ltd.) was added, and a reaction was further conducted for 4 h to obtain a second reaction solution. The second reaction solution was centrifuged at 5,000 rpm for 10 min to obtain a second precipitate, and the second precipitate was washed 3 times with pure water to finally obtain the BCDs@CaCO3-Apt. As shown in the TEM image of FIG. 1, the prepared BCDs@CaCO3-Apt has a uniform size distribution without aggregation and has a particle size of 675 nm to 925 nm. A uniform fluorescence distribution can be seen from the confocal laser scanning microscopy images. (2) Preparation of a magnetically-functionalized europium coordination polymer SI: Preparation of Fe3O4@SiO2 0.65 g of FeCh 6H2O and 0.20 g of sodium citrate were dissolved in 20 mL of ethylene glycol, then 1.20 g of NaAc was added, and stirring was conducted for 30 min to obtain a mixed solution. The mixed solution was transferred into a 50 mL Teflon liner, heated at 200°C for 10 h, and cooled to room temperature to obtain a second product. The second product was washed 3 times with each of ethanol and deionized water, and then vacuum-dried at 60°C for 6 h to obtain Fe3O4. 20 mg of the Fe3O4 was dissolved in 80 mL of ethanol and 20 mL of water, 1.25 mL of ammonia water was added, and ultrasonic dispersion was conducted for 10 min. 2.5 mL of TEOS was added, and stirring was conducted for 12 h to obtain a third product. The third product was washed 3 times with ethanol to obtain the Fe3O4@SiO2. S2: Preparation of a europium coordination polymer 0.73 g of EuC13 6H2O was dissolved in 20 mL of ethanol to obtain an ethanol solution of EuCh 6H2O. 1.30 g of BTA and 0.20 g of bpm were dissolved in 20 mL of ethanol, placed in a 50°C oil bath, and stirred magnetically to obtain a ligand solution. Then, the ethanol solution of EuCh 6H2O was slowly added dropwise to the ligand solution, a pH was adjusted with NaOH to 6 to 7, and a reaction was further conducted for 1 h. Aging, suction filtration, washing, and drying were conducted to obtain the europium coordination polymer, which was denoted as [Eu(BTA)3]2bpm. S3: Preparation of a magnetically-functionalized europium coordination polymer 20 mg of the Fe3O4@SiO2 was added to 80 mL of DMF and 20 mL of pure water, and a pH was adjusted with 0.1 mol / LHCl to 7.0. 10 mg of the [Eu(BTA)3]2bpm and 1.25 mL of ammonia water were added, and ultrasonic mixing was conducted for 10 min. 2.5 mL of TEOS was added, and a reaction was further conducted for 12 h to obtain a fourth product. The fourth product was washed 3 times with absolute ethanol and then dried to obtain Eu-Fe3O4@SiO2. 50 mg of the Eu-Fe3O4@SiO2 was dispersed in 50 mL of ultrapure water, 2 mL of APTES was added, and a reaction was conducted on a shaker for 12 h to obtain a fifth product. The fifth product was washed 3 times with each of ethanol and ultrapure water and then vacuum-dried at 50°C for 8 h. 5.0 mg of CAP-Hapten (purchased from Sangon Biotech (Shanghai) Co., Ltd.), 10 mg of sulfo-NHS, and 15 mg of EDC were dissolved in 1 mL of methanol, and a reaction was conducted for 4 h to obtain a third reaction solution. The third reaction solution was added to an MES buffer (5 mL) including 50.0 mg of the Eu-Fe3O4@SiO2, stirring was conducted for 3 h to obtain a sixth product, and the sixth product was washed to obtain the magnetically-functionalized europium coordination polymer, which was denoted as Eu-Fe3O4@SiO2-Hapten. FIG. 2 shows a transmission electron microscopy image (A) and confocal laser scanning microscopy images (B) of the Eu-Fe3O4@SiO2-Hapten. It can be seen from the transmission electron microscopy image that the europium coordination polymer prepared through self-assembly has a clear layered structure. It can be seen from the confocal laser scanning microscopy images that each layer has a strong fluorescence. (3) Preparation of the composite magnetic-assisted ratiometric fluorescent probe 30 pL of a Eu-Fe3O4@SiO2-Hapten solution and 20 pL of a BCDs@CaCO3-Apt solution were mixed to obtain the composite magnetic-assisted ratiometric fluorescent probe, which was denoted as BCDs@CaCO3-Apt / Eu-Fe3O4@SiO2-Hapten. A concentration of the BCDs@CaCO3-Apt solution was 10 mg / mL and a concentration of the Eu-Fe3O4@SiO2-Hapten solution was 10 mg / mL. Example 2 Preparation of a composite magnetic-assisted ratiometric fluorescent probe (1) Preparation of fluorescent microsphere probes BCDs@CaCO3-Apt 1.0 g of citric acid and 0.5 g of PEI were dissolved in 30 mL of ultrapure water, and ultrasonic mixing was conducted to obtain a mixture. The mixture was transferred into a 50 mL Teflon liner, heated at 220°C for 8 h, and cooled to room temperature to obtain a first product. The first product was subjected to elution with methanol, purification with a silica gel chromatographic column, and drying in a rotary evaporator to obtain a concentrated BCD solution. The concentrated BCD solution was frozen (-80°C) and then vacuum-dried to obtain BCDs. 33.4 mg of CaCh, 30 mg of CMC, and 50 mg of the BCDs were dissolved in 30 mL of water, and stirring was conducted at a constant rate for 30 min. Then 31.8 mg of Na2COs was added, and a mineralization reaction was conducted for 24 h to obtain a first reaction solution. The first reaction solution was centrifuged at 5,000 rpm for 10 min to obtain a first precipitate, and the first precipitate was washed with pure water and then vacuum-dried. 10 mg of the BCDs@CaCO3, 10 mg of sulfo-NHS, and 10 mg of EDC were dispersed in 5 mL of an MES buffer (pH = 6.2), and a reaction was conducted for 10 h. 80 pL of CAP-Apt (5.0 mg / mL) was added, and a reaction was further conducted for 4 h to obtain a second reaction solution. The second reaction solution was centrifuged at 5,000 rpm for 10 min to obtain a second precipitate, and the second precipitate was washed 3 times with pure water to finally obtain the BCDs@CaCO3-Apt. (2) Preparation of a magnetically-functionalized europium coordination polymer SI: Preparation of Fe3O4@SiO2 0.65 g of FeCh 6H2O and 0.20 g of sodium citrate were dissolved in 20 mL of ethylene glycol, then 1.20 g of NaAc was added, and stirring was conducted for 30 min to obtain a mixed solution. The mixed solution was transferred into a 50 mL Teflon liner, heated at 200°C for 10 h, and cooled to room temperature to obtain a second product. The second product was washed 3 times with each of ethanol and deionized water, and then vacuum-dried at 60°C for 6 h to obtain Fe3O4. 20 mg of the Fe3O4 was dissolved in 80 mL of ethanol and 20 mL of water, 1.25 mL of ammonia water was added, and ultrasonic dispersion was conducted for 10 min. 2.5 mL of TEOS was added, and stirring was conducted for 12 h to obtain a third product. The third product was washed 3 times with ethanol to obtain the Fe3O4@SiO2. S2: Preparation of a europium coordination polymer 0.4 g of EuC13 6H2O was dissolved in 20 mL of ethanol to obtain an ethanol solution of EuCh 6H2O. 0.8 g of BTA and 0.20 g of bpm were dissolved in 20 mL of ethanol, placed in a 50°C oil bath, and stirred magnetically to obtain a ligand solution. Then, the ethanol solution of EuCh 6H2O was slowly added dropwise to the ligand solution, a pH was adjusted with NaOH to 6 to 7, and a reaction was further conducted for 2 h. Aging, suction filtration, washing, and drying were conducted to obtain the europium coordination polymer, which was denoted as [Eu(BTA)3]2bpm. S3: Preparation of a magnetically-functionalized europium coordination polymer 20 mg of the Fe3O4@SiO2 was added to 50 mL of DMF and 20 mL of pure water, and a pH was adjusted with 0.1 mol / L HC1 to 7.0. 15 mg of the [Eu(BTA)3]2bpm and 2 mL of ammonia water were added, and ultrasonic mixing was conducted for 10 min. 3 mL of TEOS was added, and a reaction was further conducted for 15 h to obtain a fourth product. The fourth product was washed 3 times with absolute ethanol and then dried to obtain Eu-Fe3O4@SiO2. 50 mg of the Eu-Fe3O4@SiO2 was dispersed in 50 mL of ultrapure water, 5 mL of APTES was added, and a reaction was conducted on a shaker for 15 h to obtain a fifth product. The fifth product was washed 3 times with each of ethanol and ultrapure water and then vacuum-dried at 50°C for 8 h. 5.0 mg of CAP-Hapten, 15 mg of sulfo-NHS, and 20 mg of EDC were dissolved in 1 mL of methanol, and a reaction was conducted for 4 h to obtain a third reaction solution. The third reaction solution was added to an MES buffer (5 mL) including 80.0 mg of the Eu-Fe3O4@SiO2, stirring was conducted for 5 h to obtain a sixth product, and the sixth product was washed to obtain the magnetically-functionalized europium coordination polymer, which was denoted as Eu-Fe3O4@SiO2-Hapten. (3) Preparation of the composite magnetic-assisted ratiometric fluorescent probe BCDs@CaCO3-Apt / Eu-Fe3O4@SiO2-Hapten 10 pL of a Eu-Fe3O4@SiO2-Hapten solution and 30 pL of a BCDs@CaCO3-Apt solution were mixed to obtain the composite magnetic-assisted ratiometric fluorescent probe, which was denoted as BCDs@CaCO3-Apt / Eu-Fe3O4@SiO2-Hapten. A concentration of the BCDs@CaCO3-Apt solution was 10 mg / mL and a concentration of the Eu-Fe3O4@SiO2-Hapten solution was 10 mg / mL. Example 3 Preparation of a composite magnetic-assisted ratiometric fluorescent probe (1) Preparation of fluorescent microsphere probes BCDs@CaCO3-Apt 1.0 g of citric acid and 0.5 g of PEI were dissolved in 30 mL of ultrapure water, and ultrasonic mixing was conducted to obtain a mixture. The mixture was transferred into a 50 mL Teflon liner, heated at 200°C for 6 h, and cooled to room temperature to obtain a first product. The first product was subjected to elution with methanol, purification with a silica gel chromatographic column, and drying in a rotary evaporator to obtain a concentrated BCD solution. The concentrated BCD solution was frozen (-80°C) and then vacuum-dried to obtain BCDs. 33.4 mg of CaCh, 30 mg of CMC, and 50 mg of the BCDs were dissolved in 30 mL of water, and stirring was conducted at a constant rate for 30 min. Then 31.8 mg of Na2COs was added, and a mineralization reaction was conducted for 24 h to obtain a first reaction solution. The first reaction solution was centrifuged at 5,000 rpm for 10 min to obtain a first precipitate, and the first precipitate was washed with pure water and then vacuum-dried. 60 mg of the BCDs@CaCO3, 28 mg of sulfo-NHS, and 40 mg of EDC were dispersed in 5 mL of an MES buffer (pH = 6.2), and a reaction was conducted for 7 h. 120 pL of CAP-Apt (5.0 mg / mL) was added, and a reaction was further conducted for 4 h to obtain a second reaction solution. The second reaction solution was centrifuged at 5,000 rpm for 10 min to obtain a second precipitate, and the second precipitate was washed 3 times with pure water to finally obtain the BCDs@CaCO3-Apt. (2) Preparation of a magnetically-functionalized europium coordination polymer SI: Preparation of Fe3O4@SiO2 0.65 g of FeCh 6H2O and 0.20 g of sodium citrate were dissolved in 20 mL of ethylene glycol, then 1.20 g of NaAc was added, and stirring was conducted for 30 min to obtain a mixed solution. The mixed solution was transferred into a 50 mL Teflon liner, heated at 220°C for 12 h, and cooled to room temperature to obtain a second product. The second product was washed 3 times with each of ethanol and deionized water, and then vacuum-dried at 60°C for 6 h to obtain Fe3O4. 20 mg of the Fe3O4 was dissolved in 80 mL of ethanol and 20 mL of water, 1.25 mL of ammonia water was added, and ultrasonic dispersion was conducted for 10 min. 2.5 mL of TEOS was added, and stirring was conducted for 12 h to obtain a third product. The third product was washed 3 times with ethanol to obtain the Fe3O4@SiO2. S2: Preparation of a europium coordination polymer 1.4 g of EuC13 6H2O was dissolved in 20 mL of ethanol to obtain an ethanol solution of EuCh 6H2O. 1.8 g of BTA and 0.6 g of bpm were dissolved in 20 mL of ethanol, placed in a 50°C oil bath, and stirred magnetically to obtain a ligand solution. Then, the ethanol solution of EuCh 6H2O was slowly added dropwise to the ligand solution, a pH was adjusted with NaOH to 6 to 7, and a reaction was further conducted for 4 h. Aging, suction filtration, washing, and drying were conducted to obtain the europium coordination polymer, which was denoted as [Eu(BTA)3]2bpm. S3: Preparation of a magnetically-functionalized europium coordination polymer 20 mg of the Fe3O4@SiO2 was added to 130 mL of DMF and 20 mL of pure water, and a pH was adjusted with 0.1 mol / L HC1 to 7.0. 30 mg of the [Eu(BTA)3]2bpm and 3 mL of ammonia water were added, and ultrasonic mixing was conducted for 10 min. 5 mL of TEOS was added, and a reaction was further conducted for 10 h to obtain a fourth product. The fourth product was washed 3 times with absolute ethanol and then dried to obtain Eu-Fe3O4@SiO2. 80 mg of the Eu-Fe3O4@SiO2 was dispersed in 50 mL of ultrapure water, 8 mL of APTES was added, and a reaction was conducted on a shaker for 10 h to obtain a fifth product. The fifth product was washed 3 times with each of ethanol and ultrapure water and then vacuum-dried at 50°C for 8 h. 8.0 mg of CAP-Hapten, 40 mg of sulfo-NHS, and 80 mg of EDC were dissolved in 1 mL of methanol, and a reaction was conducted for 4 h to obtain a third reaction solution. The third reaction solution was added to an MES buffer (5 mL) including 100.0 mg of the Eu-Fe3O4@SiO2, stirring was conducted for 8 h to obtain a sixth product, and the sixth product was washed to obtain the magnetically-functionalized europium coordination polymer, which was denoted as Eu-Fe3O4@SiO2-Hapten. (3) Preparation of the composite magnetic-assisted ratiometric fluorescent probe BCDs@CaCO3-Apt / Eu-Fe3O4@SiO2-Hapten 10 pL of a Eu-Fe3O4@SiO2-Hapten solution and 25 pL of a BCDs@CaCO3-Apt solution were mixed to obtain the composite magnetic-assisted ratiometric fluorescent probe, which was denoted as BCDs@CaCO3-Apt / Eu-Fe3O4@SiO2-Hapten. A concentration of the BCDs@CaCO3-Apt solution was 10 mg / mL and a concentration of the Eu-Fe3O4@SiO2-Hapten solution was 10 mg / mL. Example 4 Plotting of a standard curve for CAP detection CAP standard solutions were prepared with phosphate buffered saline (PBS). 100 pL of each of the CAP standard solutions was mixed with the composite magnetic-assisted ratiometric fluorescent probe BCDs@CaCO3-Apt / Eu-Fe3O4@SiO2-Hapten in Example 1 to obtain composite systems with CAP concentrations of 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 3 ng / mL, 5 ng / mL, 7 ng / mL, 10 ng / mL, 15 ng / mL, and 20 ng / mL, respectively. The composite systems each were incubated at room temperature for 10 min to produce supernatants and precipitates. The supernatants were removed. The precipitates each were collected by a magnet and dispersed in 1 mL of PBS. Under 365 nm excitation light, fluorescence spectra of the composite systems with different CAP concentrations at 350 nm to 650 nm were recorded. As shown in FIG. 3, when no CAP is present, the composite magnetic-assisted ratiometric fluorescent probe emits both red and blue fluorescences. With the addition of CAP, a fluorescence signal change can be observed. Due to an immune response, BCDs@CaCO3-Apt preferentially binds to CAP. Under an action of a magnetic field, the red fluorescence of magnetic functionalization is retained. The blue fluorescence is removed after rinsing. Therefore, the blue fluorescence is weakened. FIG. 4 is a calibration curve produced by fitting fluorescence intensity ratios F435 / F615 at 435 nm and 615 nm to CAP concentration changes, with a corresponding function of Y = 6.663-0.304X, a correlation coefficient of R2 = 0.997, and a linear range of 0 ng / mL to 20 ng / mL. Example 5 Semi-quantitative visual detection of CAP The composite magnetic-assisted ratiometric fluorescent probe in Example 1 was mixed with CAP at different concentrations to obtain mixed solutions. The mixed solutions each were incubated for 10 min to produce supernatants and precipitates. The supernatants were removed. The precipitates each were collected by a magnet, then dispersed in PBS, and placed under an ultraviolet lamp, and corresponding color information was recorded. Color signals were mapped to the CAP concentrations, respectively, and a color change was recorded in the order from small to large. FIG. 5 shows a fluorescence color map of the composite magnetic-assisted ratiometric fluorescent probe corresponding to CAP at different concentrations. Under 365 nm ultraviolet light, it can be clearly observed by naked eyes that a fluorescence color of a mixed system changes from purple to red when different concentrations of CAP (0 ng / mL to 20 ng / mL) are added. Therefore, the composite magnetic-assisted ratiometric fluorescent probe of the present disclosure can achieve the semi-quantitative visual detection of CAP according to color information. Example 6 Detection and analysis of CAP in milk samples In order to test a quantitative analysis effect of a combination of a smart phone and the composite magnetic-assisted ratiometric fluorescent probe for actual samples, a residue in each sample to be tested was detected in this example. Specific steps were as follows: 20 pL of trichloroacetic acid (20% V / V) was added to 2 mL of a sample to obtain a mixture, and the mixture was subjected to an ultrasonic treatment for 30 min and then centrifuged at 10,000 rpm for 5 min to obtain a supernatant. The supernatant was filtered with a filter paper to obtain a CAP-containing extraction solution. The extraction solution was added to the composite magnetic-assisted ratiometric fluorescent probe in Example 1 to obtain a composite system, and the composite system was incubated for 10 min to obtain a supernatant and a precipitate. The supernatant was removed. The precipitate was collected by a magnet and then dispersed in PBS to obtain a solution. The solution was placed under a 365 nm ultraviolet lamp, and a color of the solution was observed. The solution was placed in a fluorescence spectrometer, and a fluorescence spectrum of the solution was determined under 360 nm excitation light to obtain a fluorescence intensity ratio F435 / F615. The F435 / F615 was substituted into the fitted equation in Example 4 to obtain a CAP content in the sample. An accuracy of the composite magnetic-assisted ratiometric fluorescent probe in CAP detection was investigated by a standard spiking method. Results showed that spiked CAP could be accurately measured with a high recovery rate (from 97% to 107%) and a prominent accuracy (RSD <5.2%). Example 7 Evaluation of specificity of the composite magnetic-assisted ratiometric fluorescent probe In this example, standard solutions of antibiotics (cefapirin, amoxicillin, tetracycline, enrofloxacin, and sulfamethoxazole) and inorganic ions (potassium ions, sodium ions, magnesium ions, zinc ions, carbonate ions, sulfate ions, and nitrate ions) were prepared as interfering substances to evaluate the specificity of the composite magnetic-assisted ratiometric fluorescent probe. These standard solutions all had a concentration of 1 M. Different antibiotics each were added to the composite magnetic-assisted ratiometric fluorescent probe solution in Example 1, with final antibiotic concentrations all of 10 ng / mL. The method in Example 5 was adopted for detection, and detection results were shown in FIG. 6A. FIG. 6A is a schematic diagram of selectivity of the composite magnetic-assisted ratiometric fluorescent probe in the present disclosure for the five antibiotics of cefapirin, amoxicillin, tetracycline, enrofloxacin, and sulfamethoxazole. It can be seen from this figure that a fluorescence ratio change is found merely in a solution including CAP but not in solutions including other spiked samples, indicating that the aptamer recognition has excellent selectivity and the detection of CAP in the composite system is specific and will not be interfered with by other coexisting antibiotics. CAP was mixed with different inorganic ions to obtain mixed solutions. The mixed solutions each were added to the composite magnetic-assisted ratiometric fluorescent probe solution in Example 1 to obtain composite systems each with a final inorganic ion concentration of 500 pM and a final CAP concentration of 50 pM. The method in Example 5 was adopted for detection, and detection results were shown in FIG. 6B. FIG. 6B is a schematic diagram of selectivity of the composite magnetic-assisted ratiometric fluorescent probe for potassium ions, sodium ions, magnesium ions, zinc ions, carbonate ions, sulfate ions, and nitrate ions. It can be seen from this figure that there is no significant difference between a signal of each mixed solution and a signal when CAP is present alone, indicating that the sensor has excellent selectivity for CAP and is not interfered with by other ions in a solution. In summary, a composite magnetic-assisted ratiometric fluorescent probe is successfully constructed, which can achieve the high-sensitivity and high-selectivity detection of CAP. The above examples are preferred implementations of the present disclosure, but the present disclosure is not limited to the above implementations. Any obvious improvement, substitution, or modification made by those skilled in the art without departing from the essence of the present disclosure should fall within the protection scope of the present disclosure.

Claims

1. A preparation method of a composite magnetic-assisted ratiometric fluorescent probe, characterized in that the composite magnetic-assisted ratiometric fluorescent probe is produced by compounding fluorescent microsphere probes with a magnetically-functionalized europium coordination polymer; the fluorescent microsphere probes each have a shape close to a sphere and a rough surface; the magnetically-functionalized europium coordination polymer is prepared through self-assembly, and has a shape close to a sphere and an obvious layered structure; and the fluorescent microsphere probes are uniformly distributed in different layers of the magnetically-functionalized europium coordination polymer,wherein the preparation method of the composite magnetic-assisted ratiometric fluorescent probe comprises:(1) preparation of the fluorescent microsphere probes:evenly dispersing blue carbon dots@calcium carbonate (BCDs@CaCO3), N-hydroxy sulfosuccinimide (sulfo-NHS), l-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and a chloramphenicol aptamer (CAP-Apt) in a 2-morpholinoethanesulphonic acid (MES) buffer to obtain a mixed solution, subjecting the mixed solution to a reaction at room temperature, and after the reaction, conducting centrifugation and washing to obtain the fluorescent microsphere probes, denoted as BCDs@CaCO3-Apt;(2) preparation of the magnetically-functionalized europium coordination polymer:SI, preparation of a europium coordination polymer:dissolving benzoyl(trifluoroacetyl)methane (BTA) and 2,2'-bipyrimidine (bpm) in an ethanol solution, slowly adding an ethanol solution of europium chloride hexahydrate, and thoroughly mixing to obtain a mixed reaction system; adjusting a pH of the mixed reaction system to 6 to 7, and conducting an aging reaction; and after the aging reaction, conducting suction filtration, washing, and drying to obtain the europium coordination polymer, denoted as [Eu(BTA)3]2bpm; andS2, preparation of the magnetically-functionalized europium coordination polymer:thoroughly mixing Fe3O4@SiO2 with N,N-dimethylformamide (DMF), and adjusting a pH to 7.0; adding the [Eu(BTA)3]2bpm and ammonia water, and conducting ultrasonic mixing fully; adding tetraethyl orthosilicate (TEOS), and conducting a first stirring-based reaction; after the first stirring-based reaction, conducting centrifugation, washing, and drying to obtain Eu-Fe3O4@SiO2;adding a 3-aminopropyltri ethoxy silane (APTES) solution to a Eu-Fe3O4@SiO2 solution to allow a reaction, and after the reaction, conducting washing and drying to obtain Eu-Fe3O4@SiO2-NH2;mixing a chloramphenicol hapten (CAP-Hapten), sulfo-NHS, and EDC in a methanol solution, adding an MES buffer of the Eu-Fe3O4@SiO2-NH2, and conducting a second stirring-based reaction; and after the second stirring-based reaction, conducting separation and washing to finally obtain the magnetically-functionalized europium coordination polymer, denoted as Eu-Fe3O4@SiO2-Hapten; and(3) preparation of the composite magnetic-assisted ratiometric fluorescent probe:thoroughly mixing a BCDs@CaCO3-Apt solution with a Eu-Fe3O4@SiO2-Hapten solution to obtain a composite magnetic-assisted ratiometric fluorescent probe solution, denoted as BCDs@CaCO3-Apt / Eu-Fe3O4@SiO2-Hapten.

2. The preparation method of the composite magnetic-assisted ratiometric fluorescent probe according to claim 1, characterized in that in the step (1), the BCDs@CaCO3, the sulfo-NHS, and the EDC are in a mass ratio of (5 to 15):(2 to 8):(3 to 12);the reaction at room temperature is conducted for 4 h to 10 h; anda final concentration of the CAP-Apt in the mixed solution is 0.1 mg / mL to 1 mg / mL.

3. The preparation method of the composite magnetic-assisted ratiometric fluorescent probe according to claim 1, characterized in that in the SI, the ethanol solution of the europium chloride hexahydrate, the BTA, and the bpm are in a mass ratio of (2 to 8):(5 to 10):(1 to 4);in the mixed reaction system, a total volume of ethanol is the same as a volume sum of the ethanol solution of the europium chloride hexahydrate, the BTA, and the bpm; andthe aging reaction is conducted for 1 h to 4 h.

4. The preparation method of the composite magnetic-assisted ratiometric fluorescent probe according to claim 1, characterized in that when the Eu-Fe3O4@SiO2 is prepared in the S2:the Fe3O4@SiO2, the DMF, the [Eu(BTA)3]2bpm, the ammonia water, and the TEOS are in an amount ratio of (20 to 60) mg : (80 to 240) mL : (10 to 30) mg : (1.25 to 3.75) mL : (1 to 5) mL; andthe first stirring-based reaction is conducted as follows: ultrasonically stirring for 10 min to 20 min, and allowing a reaction at room temperature for 10 h to 15 h.

5. The preparation method of the composite magnetic-assisted ratiometric fluorescent probe according to claim 1, wherein when the Eu-Fe3O4@SiO2-NH2 is prepared in the S2:a volume ratio of the APTES solution to the Eu-Fe3O4@SiO2 solution is (10 to 50):(1 to 5); a solvent for the APTES solution is absolute ethanol and a solvent for the Eu-Fe3O4@SiO2 solution is pure water; andthe reaction is conducted at room temperature for 10 h to 15 h.

6. The preparation method of the composite magnetic-assisted ratiometric fluorescent probe according to claim 1, characterized in that when the Eu-Fe3O4@SiO2-Hapten is prepared in the S2:the CAP-Hapten, the sulfo-NHS, the EDC, and the Eu-Fe3O4@SiO2 are in a mass ratio of (1 to 5):(2 to 10):(3 to 15):(10 to 50); andthe second stirring-based reaction is conducted under stirring for 4 h to 8 h.

7. The preparation method of the composite magnetic-assisted ratiometric fluorescent probe according to claim 1, characterized in that in the step (3), a volume ratio of the BCDs@CaCO3-Apt solution to the Eu-Fe3O4@SiO2-Hapten solution is (1 to 6):(1 to 9); andthe BCDs@CaCO3-Apt solution and the Eu-Fe3O4@SiO2-Hapten solution have a same concentration.

8. A use of a composite magnetic-assisted ratiometric fluorescent probe prepared by the preparation method according to claims 1 to 7 in visual detection of chloramphenicol (CAP).

9. The use according to claim 8, characterized in that the visual detection refers to semi-quantitative detection or quantitative detection.

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