Preparation method and application of ciprofloxacin ratiometric fluorescent probe

Abstract

The invention belongs to the technical field of composite material preparation and food safety detection, and particularly relates to a preparation method and application of a ciprofloxacin ratiometric fluorescent probe based on perovskite quantum dot-carbon dot molecular imprinting. The ratiometric fluorescent probe is prepared by the following steps: firstly, preparing perovskite quantum dots (PQDs) and carbon dots (CDs); then, compounding the two fluorescent materials through a sol-gel method to form a PQDs-CDs composite material; and finally, synthesizing the ratio type fluorescent probe PQDs-CDs (at) MIP by adopting a molecular imprinting technology and combining specific recognition of a target molecule ciprofloxacin (CIP). The preparation method of the material is simple, the synthesis environment is friendly, the prepared composite material combines the advantages of the perovskite quantum dots, the carbon dots and the molecularly imprinted polymer, and the recognition capability on target molecules can be efficiently improved. Compared with a traditional detection method, the method not only has high sensitivity and selectivity, but also provides an efficient and economical solution for detection of CIP in various aquatic products, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of composite material preparation and food safety detection, and specifically relates to a preparation method and application of a ciprofloxacin ratiometric fluorescent probe based on perovskite quantum dot-carbon dot molecular imprinting. Background Art

[0002] Ciprofloxacin (CIP) is a broad-spectrum antibiotic widely used in clinical treatment and treatment of bacterial infections in animal husbandry. Its large-scale use causes residues in the environment and food, causing potential drug resistance problems, and has attracted widespread attention worldwide. Existing detection methods mainly include large-scale instrument detection represented by high-performance liquid chromatography and rapid screening detection represented by enzyme-linked immunosorbent assay. Instrument detection methods can provide high-precision test results, but most of them are costly, cumbersome to operate or take a long time to detect. For enzyme-linked immunosorbent assay, the accuracy of the test results depends largely on the performance of the enzyme, and the enzyme is more easily affected by the use environment. Therefore, the development of a rapid, sensitive and accurate ciprofloxacin detection method is of great significance for protecting the environment and food safety.

[0003] In view of the rapid response and high sensitivity of fluorescence sensing, fluorescent probes for ciprofloxacin have been established. Among them, probes that rely on a single wavelength are easily affected by factors such as light source fluctuations and solution environment, making it difficult to achieve ideal detection stability and accuracy. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for preparing a ratiometric fluorescent probe of ciprofloxacin, that is, using two heterogeneous fluorescent materials, carbon dots and perovskite quantum dots, to construct a ratiometric fluorescent probe with dual signal response.

[0005] The present invention also provides the application of the above-mentioned ratio-type fluorescent probe based on perovskite quantum dots-carbon dots molecular imprinting fluorescent composite material in the detection of CIP. The ratio-type fluorescent probe has shown significant advantages in the detection of CIP. By utilizing the different fluorescence response characteristics of carbon dots and perovskite quantum dots in the presence of CIP, rapid and highly sensitive detection of CIP can be achieved. In addition, the probe is also combined with molecular imprinting technology, has a high degree of selectivity for CIP, and can effectively avoid the influence of interfering substances in complex samples, thereby ensuring the accuracy of the detection results. Therefore, this technology can be widely used in the detection of CIP in foods such as aquatic products, and also provides feasible ideas and technologies for the detection of other harmful substances in food.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: The present invention provides a method for preparing a ciprofloxacin ratiometric fluorescent probe, comprising the following steps: (1) Preparation of carbon dots (CDs) The reaction solvent was heated under a nitrogen atmosphere with strong stirring, and anhydrous citric acid was immediately added, and the reaction was kept warm. After the reaction was completed, the reaction solvent was cooled to room temperature, washed, filtered, and dispersed in anhydrous ethanol to obtain a CDs ethanol suspension; (2) Preparation of perovskite quantum dots (PQDs) Preparation of the precursor: heating the three reaction solvents under a nitrogen atmosphere to obtain a cesium oleate precursor; Preparation of PQDs: Lead bromide and octadecene were heated to react in a nitrogen atmosphere, and then oleic acid and APTES were added, and then the temperature was raised and cesium oleate precursor was quickly added. After cooling, the mixture was stirred in a water bath for hydrolysis. After the hydrolysis was completed, the mixture was centrifuged to remove the supernatant, washed with n-hexane, vacuum dried, and ground to obtain PQDs; (3) Preparation of PQDs-CDs@MIP The template molecule was dissolved in octadecene by the sol-gel method, and a CDs ethanol suspension, PQDs and functional monomers were added to carry out a prepolymerization reaction. Then, after adding the cross-linking agent, stirring was continued for a period of time. The product was collected by centrifugation, washed with an eluent, and finally vacuum dried to obtain PQDs-CDs@MIP.

[0007] Furthermore, in step (1), the reaction solvent is 3-(2-aminoethylamino)propylmethyldimethoxysilane (AEAMPS); the temperature is raised to 235-245°C, and anhydrous citric acid is immediately added; the insulation reaction time is 1-2 minutes; and 0.5 g of anhydrous citric acid is added to every 10 mL of 3-(2-aminoethylamino)propylmethyldimethoxysilane.

[0008] Furthermore, in step (2), the three reaction solvents are cesium carbonate (CsCO3), oleic acid and octadecene (ODE); the ratio of cesium carbonate (CsCO3), oleic acid and octadecene (ODE) is:; the heating reaction process is: at a speed of 500r / min, the temperature is increased to 120-125°C and maintained for 1h; then the temperature is continued to be increased to 140-145°C and the reaction is kept warm for 2h.

[0009] Furthermore, in step (2), the ratio of lead bromide to octadecene is 0.138 g:10 ml; the temperature is raised to 120° C. at a speed of 500 r / min and kept for 1 hour; 50 μL of oleic acid, 1 ml of APTES and 1 mL of cesium oleate precursor are added to every 10 mL of octadecene.

[0010] Furthermore, in step (2), the hydrolysis is carried out by stirring for 3 hours in a water bath at 20-25°C; the centrifugation is carried out at 8000 r / min for 8 minutes; and the vacuum drying temperature is 60°C.

[0011] Furthermore, in step (3), the template molecule is CIP; the functional monomer is APTES; the cross-linking agent is TMOS; the eluent is a mixture of n-hexane and acetic acid; the volume ratio of n-hexane to acetic acid is 8:2; and the vacuum drying temperature is 60°C.

[0012] Preferably, in step (3), the ratio of the template molecule, the functional monomer and the cross-linking agent is 1:3-6:8-32; the ratio of the template molecule, the CDs ethanol suspension and the PQDs is 1 mmmol:400-700 μL:30 mg.

[0013] The present invention also provides a ciprofloxacin ratiometric fluorescent probe prepared by the preparation method.

[0014] The present invention also provides the use of the above-mentioned ciprofloxacin ratiometric fluorescent probe in analyzing and detecting CIP, which is characterized by comprising the following steps: (1) Add PQDs-CDs@MIP to the sample extract and shake at room temperature to obtain the sample to be tested; (2) The sample to be tested is detected and analyzed using a fluorescence spectrophotometer.

[0015] Preferably, the solid-liquid ratio of the PQDs-CDs@MIP and the sample extract is 2 mg:3 mL; the room temperature oscillation time is 25 min; the conditions for the fluorescence spectrophotometer detection and analysis are: the excitation wavelength is 365 nm, the emission wavelength is 400~700 nm, and the slit width is 5 nm.

[0016] The present invention adopts surface free radical polymerization imprinting technology, and introduces functional group-modified carbon dots to combine with perovskite materials to prepare a perovskite quantum dot-carbon dot molecular imprinting fluorescent composite ratio fluorescent probe. The probe uses the excellent fluorescence properties of carbon dots to improve the sensitivity and stability of the material, and at the same time enhances the response intensity of the fluorescent signal through the photoelectric properties of the perovskite material. In the molecular imprinting process, the introduction of the perovskite material can effectively improve the selectivity of the molecular imprinting layer to the target molecule, and form specific molecular recognition sites through molecular imprinting technology, further enhancing the recognition ability of CIP. The material can not only highly selectively identify CIP, but also avoid interfering substances in complex samples, providing more accurate and sensitive detection results.

[0017] The beneficial effects of the present invention are: (1) The preparation method of the ratiometric fluorescent probe of the perovskite quantum dot-carbon dot molecular imprinted fluorescent composite material provided by the present invention is simple and the synthesis process is environmentally friendly. The effective combination of carbon dots and perovskite quantum dots enhances the fluorescent properties of the material, and molecular imprinting polymerization improves the selectivity of molecular recognition. Therefore, the material exhibits excellent properties such as high selectivity and high sensitivity when identifying the target.

[0018] (2) The ratiometric fluorescent probe of the perovskite quantum dot-carbon dot molecular imprinted fluorescent composite material prepared by the present invention can be directly applied to the detection of CIP in complex matrices, achieving highly selective and sensitive analytical detection of CIP in food samples. The material is easy to operate in rapid detection and has a short detection cycle, which greatly shortens the time required for traditional detection methods. In addition, the technology has high detection sensitivity and can accurately identify and quantitatively analyze CIP in complex backgrounds, with a minimum detection limit of 0.005 μmol / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the optimization diagram of the amount of CDs added in the PQDs-CDs@MIP material; Figure 2 This is the optimized ratio diagram between template molecules, functional monomers and cross-linking agents in PQDs-CDs@MIP materials; Figure 3 Transmission electron microscopy of PQDs-CDs@MIP material; Figure 4 XRD pattern of PQDs-CDs@MIP material; Figure 5 The fluorescence selectivity of PQDs-CDs@MIP material to CIP; Figure 6 This is the standard curve of the fluorescence response of PQDs-CDs@MIP to CIP solution. DETAILED DESCRIPTION

[0020] In order to make the above features and advantages of the present invention clearer and easier to understand, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0021] Unless otherwise specified, the reagents involved in the following embodiments are commercially available and were not processed before use.

[0022] Example 1 The preparation method based on PQDs-CDs@MIP material includes the following steps: (1) Accurately measure 10 mL of 3-(2-aminoethylamino)propylmethyldimethoxysilane (AEAMPS) and place it in a three-necked round-bottom flask. Connect N2 to deoxygenate for 10 min. After deoxygenation, quickly move the three-necked round-bottom flask to a heating mantle, connect a condenser and connect N2 to keep the N2 atmosphere. Raise the reaction temperature to 240°C at 500 r / min. After the solution is heated to 240°C, quickly add 0.5 g of anhydrous citric acid and continue the reaction for 1 min. After the reaction is completed, cool to room temperature and finally vortex wash and purify with petroleum ether until the upper liquid is clear. Filter with a 0.22 μm filter membrane and disperse it in 100 mL of anhydrous ethanol to obtain a CDs ethanol suspension. Seal and store in a dark place at 4°C for later use.

[0023] (2) Add 0.8 g of cesium carbonate (CsCO3), 2.5 mL of oleic acid and 30 mL of octadecene (ODE) to a 100 mL three-necked round-bottom flask and connect a double-row tube. First, screw the double-row tube to the vacuum tube, evacuate for 10 min, and then screw it to the N2 tube. The subsequent reaction must always maintain the N2 atmosphere. Raise the reaction temperature to 120°C at a speed of 500 r / min, maintain for 1 h, then raise the temperature to 150°C and maintain for 2 h to obtain a cesium oleate precursor. The prepared cesium oleate precursor is sealed and stored in a dry place.

[0024] 0.138 g of lead bromide (PbBr2) and 10 mL of octadecene were added to a 100 mL three-necked round-bottom flask, and a double-row tube was connected externally. The double-row tube was first screwed to the vacuum tube, vacuumed for 10 min, and then screwed to the N2 tube to keep the reaction in a N2 atmosphere. The reaction temperature was raised to 120°C at a speed of 500 r / min and maintained for 1 h. Then 50 μL of oleic acid and 1 mL of APTES were injected, and then the temperature was raised to 140°C, and 1 mL of cesium oleate precursor (preheated to 100°C) was quickly injected. After 5 s, the reaction was cooled to room temperature with an ice water bath. Finally, the reaction was stirred in a water bath at 25°C for 3 h for hydrolysis. After the hydrolysis was completed, it was centrifuged at 8000 r / min for 8 min, the supernatant was removed, and it was washed three times with n-hexane, vacuum dried at 60°C for 12 h, and finally ground with an agate mortar to obtain PQDs.

[0025] (3) Using the sol-gel method, 1mmmol of the template molecule CIP was dissolved in 15 mL of octadecene, and 500μLCDs and 30mg of PQDs were added as two fluorescent signal substances, 5mmmol APTES was used as a functional monomer, and prepolymerization was stirred at 25°C for 30min. 20mmmol TMOS was used as a crosslinker, and the mixture was stirred at 25°C for 15h. The product was collected by centrifugation at 8000 r / min for 8min. The product was washed several times with an eluent of anhydrous ethanol: acetic acid (8:2, v / v) and dried in vacuum at 60°C to obtain PQDs-CDs@MIP. The above steps were repeated without adding the template molecule CIP to obtain PQDs-CDs@NIP.

[0026] Example 2 The amount of CDs added and the ratio between template molecules, functional monomers and cross-linking agents were optimized and analyzed. Figure 1 As shown in the figure, when the amount of CDs is 500 μL, the sensitivity of the fluorescent probe is the highest, so 500 μL is selected as the optimal amount of CDs added; Figure 2 As shown, when the ratios of the three are 1:3:8, 1:4:16, 1:5:20, and 1:6:32, respectively, PQDs-CDs@MIP and PQDs-CDs@NIP respond to different concentrations of CIP, and the molecular imprinting probe synthesized in a ratio of 1:5:20 has the best fluorescence response to CIP, with the highest imprinting factor of 2.5, so the ratio of 1:5:20 was selected to synthesize the polymer.

[0027] Effect Example 1. Study on the properties of PQDs-CDs@MIP materials In order to further understand the properties of the prepared materials, the properties of the PQDs-CDs@MIP material prepared in Example 1 were studied.

[0028] Figure 3 The transmission electron micrographs of PQD and PQDs-CDs@MIP materials are shown in Figure 1. As shown in the figure, PQDs have a cubic structure, and PQDs-CDs@MIP presents a distinct core-shell structure, with the core being aggregated PQDs and the outer layer being a molecular imprinting layer.

[0029] Figure 4 Figure 2 is the XRD pattern of PQDs-CDs@MIP material. As shown in the figure, after PQDs were embedded in the MIP imprinting layer, the characteristic diffraction peaks of PQDs basically disappeared in the MIP curve due to the modification of the imprinting shell, indicating the successful synthesis of PQDs-CDs@MIP.

[0030] Figure 5This is a fluorescence selectivity experiment of PQDs-CDs@MIP material to the same concentration of CIP and its structural analogs enrofloxacin, ofloxacin, levofloxacin, and pefloxacin. As shown in the figure, the fluorescence responses of PQDs-CDs@MIP and PQDs-CDs@NIP to CIP are higher than those of the other substances, and the fluorescence response of PQDs-CDs@MIP to CIP is higher than that of PQDs-CDs@NIP, indicating that PQDs-CDs@MIP can specifically recognize CIP.

[0031] Figure 6 Figure 2 is the standard curve of the fluorescence response of PQDs-CDs@MIP material to different concentrations of CIP. As shown in the figure, when the concentration of CIP is in the range of 0.01-30 μmol / L, the fluorescence response signal F of PQDs-CDs@MIP and PQDs-CDs@NIP is CDs / F PQDs It showed a good linear relationship with the CIP concentration; the minimum detection limit of this method was 0.005 μmol / L.

[0032] 2. Application of PQDs-CDs@MIP materials in CIP analysis and detection The prepared polymer CDs@RAM-MIP was added to the sample extract at a solid-liquid ratio of 2 mg:3 mL, and shaken at room temperature for 25 minutes before use for fluorescence spectrophotometer analysis. This method was applied to the determination of CIP in actual samples and compared with the results of HPLC detection. The results are shown in Table 1.

[0033] Table 1 Application of PQDs-CDs@MIP materials in fluorescence sensing of CIP

Claims

1. A method for preparing a ciprofloxacin ratiometric fluorescent probe, characterized in that: The following steps are involved: (1) Preparation of carbon dots (CDs) The reaction solvent was heated under a nitrogen atmosphere with strong stirring, and anhydrous citric acid was immediately added, and the reaction was kept warm. After the reaction was completed, the reaction solvent was cooled to room temperature, washed, filtered, and dispersed in anhydrous ethanol to obtain a CDs ethanol suspension; (2) Preparation of perovskite quantum dots (PQDs) Preparation of the precursor: heating the three reaction solvents under a nitrogen atmosphere to obtain a cesium oleate precursor; Preparation of PQDs: Lead bromide and octadecene were heated to react in a nitrogen atmosphere, and then oleic acid and APTES were added, and then the temperature was raised and cesium oleate precursor was quickly added. After cooling, the mixture was stirred in a water bath for hydrolysis. After the hydrolysis was completed, the mixture was centrifuged to remove the supernatant, washed with n-hexane, vacuum dried, and ground to obtain PQDs; (3) Preparation of PQDs-CDs@MIP Using the sol-gel method, the template molecule was dissolved in octadecene, and a CDs ethanol suspension, PQDs and functional monomers were added to perform a prepolymerization reaction. After adding the cross-linking agent, stirring was continued for a period of time. The product was collected by centrifugation, washed with an eluent, and finally dried in vacuum to obtain PQDs-CDs@MIP.

2. The preparation method according to claim 1, characterized in that: In step (1), the reaction solvent is 3-(2-aminoethylamino)propylmethyldimethoxysilane (AEAMPS); the temperature is raised to 235-245°C, and anhydrous citric acid is immediately added; the insulation reaction time is 1-2 minutes; 0.5 g of anhydrous citric acid is added to every 10 mL of 3-(2-aminoethylamino)propylmethyldimethoxysilane.

3. The preparation method according to claim 1 or 2, characterized in that: In step (2), the three reaction solvents are cesium carbonate (CsCO3), oleic acid and octadecene (ODE); the ratio of cesium carbonate (CsCO3), oleic acid and octadecene (ODE) is:; the heating reaction process is: at a speed of 500r / min, the temperature is increased to 120-125°C and maintained for 1h; then the temperature is continued to be increased to 140-145°C and the reaction is kept warm for 2h.

4. The preparation method according to any one of claims 1 to 3, characterized in that In step (2), the ratio of lead bromide to octadecene is 0.138 g:10 ml; the temperature is raised to 120° C. at a speed of 500 r / min and kept for 1 hour; 50 μL of oleic acid, 1 ml of APTES and 1 mL of cesium oleate precursor are added to every 10 mL of octadecene.

5. The preparation method according to claim 4, characterized in that: In step (2), the hydrolysis is carried out by stirring for 3 hours in a water bath at 20-25°C; the centrifugation is carried out at 8000 r / min for 8 minutes; and the vacuum drying temperature is 60°C.

6. The preparation method according to any one of claims 1 to 5, characterized in that: In step (3), the template molecule is CIP; the functional monomer is APTES; the cross-linking agent is TMOS; the eluent is a mixture of n-hexane and acetic acid; the volume ratio of n-hexane to acetic acid is 8:2; and the vacuum drying temperature is 60°C.

7. The preparation method according to claim 6, characterized in that: In step (3), the ratio of the template molecule, the functional monomer and the cross-linking agent is 1:3-6:8-32; the ratio of the template molecule, the CDs ethanol suspension and the PQDs is 1 mmmol:400-700 μL:30 mg.

8. A ciprofloxacin ratiometric fluorescent probe prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the ciprofloxacin ratiometric fluorescent probe as claimed in claim 8 in analyzing and detecting CIP, characterized in that: The following steps are involved: (1) Add PQDs-CDs@MIP to the sample extract and shake at room temperature to obtain the sample to be tested; (2) The sample to be tested is detected and analyzed using a fluorescence spectrophotometer.

10. The use according to claim 9, characterized in that: The solid-liquid ratio of the PQDs-CDs@MIP and the sample extract is 2 mg:3 mL; the room temperature oscillation time is 25 min; the conditions for the fluorescence spectrophotometer detection and analysis are: the excitation wavelength is 365 nm, the emission wavelength is 400-700 nm, and the slit width is 5 nm.

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