Preparation method and application of PQDs-LMOF-based enrofloxacin 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 an enrofloxacin ratiometric fluorescent probe based on PQDs-LMOF. According to the method, perovskite quantum dots (PQDs) grow in situ into a light-emitting metal organic framework (LMOF), and a molecular imprinting technology is combined, so that a novel ratio type fluorescent probe, namely the perovskite quantum dot-light-emitting metal organic framework molecular imprinting fluorescent probe (PQDs-LMOF (at) MIP) is constructed. The probe can form a specific molecular recognition site for enrofloxacin (ENR) molecules, so that high-sensitivity and high-selectivity detection of ENR in aquatic products is realized; by adjusting the optical characteristics of the perovskite quantum dots and the light-emitting metal organic frame, the accurate detection of the ENR is realized by means of the change of the fluorescence signal ratio. The material prepared by the invention can be directly used for detection and analysis of the ENR in a complex aquatic product sample, and high-selectivity and high-sensitivity analysis and detection of the ENR in the aquatic product are realized; the operation is simple and convenient, the detection period is short, and the detection time is greatly shortened; according to the invention, the detection sensitivity is high, and the lowest detection limit is 0.026 [mu] mol / L.

Description

Technical Field

[0001] The present invention belongs to the technical field of composite material preparation and food safety detection, and specifically relates to a preparation method and application of an enrofloxacin ratiometric fluorescent probe based on perovskite quantum dots-luminescent metal organic framework (PQDs-LMOF). Background Art

[0002] As global food safety issues have received increasing attention, the detection of drug residues, especially antibiotics, has become an important topic in the field of food safety testing. Enrofloxacin (ENR), a fluoroquinolone antibiotic commonly used in animal and human treatment, may remain in the environment, food, and water sources due to its widespread use, posing a potential threat to the ecological environment and human health. Therefore, the development of a sensitive and efficient detection technology to monitor ENR residues in food has become an urgent problem to be solved. Existing ENR detection methods mainly include traditional methods such as high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). Although these methods have high detection accuracy, they cannot meet the requirements of sensitive and efficient detection in food safety testing due to their complex operating steps, high equipment costs, and long detection time. Summary of the invention

[0003] In view of the problems existing in the prior art, the present invention proposes a method for preparing an enrofloxacin ratiometric fluorescent probe based on perovskite quantum dots-luminescent metal organic framework (PQDs-LMOF), which improves the sensitivity and selectivity of detection of ENR. This new molecular imprinted fluorescent probe can not only efficiently and quickly identify and detect ENR residues, but also overcomes the defects of traditional methods and has broad application prospects.

[0004] The present invention also provides the application of the above-mentioned PQDs-LMOF-based enrofloxacin ratiometric fluorescent probe in ENR sensing detection. Based on the ratio of the fluorescence intensity between PQDs and LMOF, a highly sensitive and highly selective ENR detection method is constructed, the linear range of the method is 0.05-50 μmol / L, the minimum detection limit is 0.026 μmol / L, and the method is used for the determination of ENR in a variety of aquatic products.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: The present invention provides a method for preparing an enrofloxacin ratiometric fluorescent probe based on PQDs-LMOF, comprising the following steps: (1) Preparation of LMOF Dissolve europium chloride hexahydrate (EuCl3·6H2O) and succinimide (SI) in appropriate amount of deionized water, stir and ultrasonically treat to obtain Eu 3+ solution and SI solution; then, citric acid was added to Eu 3+ The solution was stirred evenly to obtain Eu-CA solution; the SI solution was added to the Eu-CA solution and continued to be stirred, and then the ethanol solution of thenoyl trifluoroacetone was added and stirred at a suitable temperature for a period of time. Subsequently, the pH value was adjusted, and the stirring was continued and maintained for a period of time, and then cooled to room temperature, centrifuged to separate the precipitate, washed and vacuum dried to obtain LMOF; (2) Preparation of PQDs-LMOF Cesium carbonate (CsCO3), oleic acid and octadecene (ODE) are deoxygenated with N2 and then moved to a heating mantle, and the temperature is raised in stages under a nitrogen atmosphere to carry out a heat preservation reaction to obtain a cesium oleate precursor; Lead bromide and octadecene are evacuated and then connected to nitrogen. After heating to a certain temperature, oleic acid and APTES are added, and the temperature is continued to rise to disperse LMOF in the lead source. Cesium oleate precursor is quickly added, and after a short time, it is cooled in an ice water bath, hydrolyzed, centrifuged, washed, and vacuum dried to obtain PQDs-LMOF. (3) Preparation of PQDs-LMOF@MIP The template molecule was dissolved in n-hexane, functional monomers and PQDs-LMOF were added, a cross-linker was added after prepolymerization and stirred overnight, the product was collected by centrifugation and washed with a mixed solvent of n-hexane and acetic acid, and finally dried under vacuum to obtain PQDs-LMOF@MIP; after removing the template molecule, the above steps were repeated to obtain PQDs-LMOF@NIP.

[0006] Preferably, in step (1), the molar ratio of europium chloride hexahydrate to succinimide is 1:3 mmol; the molar ratio of europium chloride hexahydrate to citric acid is 1:0.5; the molar ratio of succinimide to 2-thenoyltrifluoroacetone (TTA) is 3:0.75 mmol, and the mixture is stirred at 50°C for 15 min; the pH is adjusted to 9 using 1 mol / L sodium bicarbonate solution; and the condition of heat preservation and stirring is stirring at 50°C for 30 min.

[0007] Preferably, in step (2), the ratio of cesium carbonate (CsCO3), oleic acid and octadecene (ODE) is 0.8 g:2.5 mL:30 mL; the ratio of lead bromide, octadecene (ODE), oleic acid and APTES is 0.138 g:10 mL:50 μL:1 mL; the mass ratio of LMOF and lead bromide is 80:0.13-0.14.

[0008] Preferably, in step (2), the stepwise heating is firstly heating to 120°C and maintaining for 1 hour; then heating to 140°C and reacting for 2 hours.

[0009] Preferably, in step (2), after adding oleic acid and APTES, the reaction temperature is raised to 120°C at a rotation speed of 500 r / min and maintained for 1 h; then the temperature is raised to 140°C; the hydrolysis is carried out under stirring in an oil bath at 25°C for 3 h; and the vacuum drying temperature is 60°C.

[0010] Preferably, in step (3), the molar ratio of the template molecule, the functional monomer and the cross-linking agent is 1:3-5:8-32; the ratio of the template molecule to PQDs-LMOF is 1 mmol:200-1000 mg; and the eluent is composed of n-hexane and acetic acid in a volume ratio of 8:2.

[0011] Preferably, the template molecule is ENR; the functional monomer is APTES; the cross-linking agent is TMOS; and the vacuum drying temperature is 60°C.

[0012] The present invention also provides an application of the enrofloxacin ratiometric fluorescent probe prepared by the above preparation method in analyzing and detecting ENR, which is characterized by comprising the following steps: (1) Add the prepared polymer PQDs-LMOF@MIP into the sample extract according to the material-liquid ratio, shake at room temperature, and use for analysis and detection.

[0013] (2) The sample to be tested is detected and analyzed using a fluorescence spectrophotometer.

[0014] Preferably, the ratio of the PQDs-LMOF@MIP to the sample extract is 2 mg:3 mL; and the shaking time at room temperature is 15 min.

[0015] Preferably, the conditions for the fluorescence spectrophotometer detection and analysis are: an excitation wavelength of 365 nm, an emission wavelength of 400-700 nm, and a slit width of 5 nm.

[0016] The present invention in-situ grows PQDs with excellent optical properties into highly stable LMOF, combines molecular imprinting technology with the sol-gel method to construct a new ratiometric molecular imprinting fluorescent probe - a new ratiometric fluorescent probe of perovskite quantum dots-luminescent metal organic framework (PQDs-LMOF) (PQDs-LMOF@MIP), so as to achieve sensitive detection of ENR in aquatic products.

[0017] The beneficial effects of the present invention are: (1) The preparation method of the perovskite quantum dot-luminescent metal organic framework (PQDs-LMOF) ratiometric fluorescent probe (PQDs-LMOF@MIP) provided by the present invention is simple, the synthesis conditions are relatively mild, and no complex synthesis environment is required. By combining molecular imprinting technology and the characteristics of fluorescent materials, the material has excellent characteristics such as high selectivity, high sensitivity, and rapid response to ENR.

[0018] (2) The PQDs-LMOF@MIP material prepared by the present invention can be directly applied to the detection and analysis of ENR in complex matrices, significantly improving the selectivity and sensitivity of ENR in samples such as food and environment. The material has the characteristics of high selectivity, low interference, and rapid response, and can complete efficient and accurate detection in a short time. Its minimum detection limit is 0.026 μmol / L, and it is easy to operate and promote and apply. Through the ratiometric fluorescence signal response, the technology of the present invention can effectively reduce the influence of interfering substances in the sample on the detection results, ensuring the accuracy and reliability of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Transmission electron microscopy of PQDs-LMOF@MIP material; Figure 2 X-ray diffraction pattern of PQDs-LMOF@MIP material; Figure 3 The fluorescence selectivity of PQDs-LMOF@MIP material to ENR; Figure 4 This is the standard curve of the fluorescence response of PQDs-LMOF@MIP to ENR 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-LMOF@MIP material includes the following steps: (1) Disperse 1 mmol europium chloride hexahydrate (EuCl3·6H2O) and 3 mmol succinimide (SI) in 2 mL and 4 mL deionized water, respectively, stir at room temperature for 20 min, and then ultrasonicate for 20 min. 3+0.2 ml of 2.5 mol / L citric acid (CA) solution was added to the solution and stirred at room temperature for 20 min to obtain Eu-CA solution. After that, SI solution was added to Eu-CA solution and continued to stir for 15 min. Then 0.75 mmol of 2-thenoyltrifluoroacetone (TTA) ethanol solution (0.4 mol / L) was added and stirred at 50 °C for 15 min. Subsequently, 1 mol / L sodium bicarbonate (NaHCO3) was slowly added to the solution until pH = 9 was reached. The reaction mixture was kept at 50 °C with stirring for 30 min and then cooled to room temperature. The europium complex was precipitated, centrifuged at 10000 r / min for 10 min, and washed three times with deionized water. Finally, the complex was dried under vacuum at room temperature.

[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 the temperature was immediately raised to 140°C. A certain mass of LMOF was evenly dispersed in the lead source, and 1 mL of cesium oleate precursor (preheated to 100°C) was quickly injected. After 5 s, the reaction was cooled to room temperature in 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.

[0025] (3) Using sol-gel technology, the template molecule ENR was dissolved in 10 mL of n-hexane, and then the functional monomer APTES and the fluorescent response material PQDs-LMOF were added, prepolymerized at 25°C for 30 min, and then the crosslinker TMOS was added, stirred overnight at 25°C, and centrifuged at 8000 r / min for 8 min to collect the product. The product needed to be washed several times with the eluent n-hexane: acetic acid (8:2, v:v), and finally dried in vacuum at 60°C to obtain PQDs-LMOF@MIP. Without the template molecule ENR, the above steps were repeated to obtain PQDs-LMOF@NIP.

[0026] Example 2 The amount of LMOF added during the in-situ growth preparation of the fluorescent response material PQDs-LMOF was optimized and analyzed. 30 mg of PQDs-LMOF materials were prepared with different amounts of LMOF, and the composite sensing material PQDs-LMOF@MIP was prepared when the template molecule, functional monomer and cross-linking agent were 0.1mmol: 0.4mmol: 1.6mmol respectively. The ratio of the fluorescence quenching intensity of PQDs-LMOF@MIP and PQDs-LMOF@NIP by CIP solution (imprinting factor) was used as a measurement indicator. The specific dosage relationship and imprinting factor are shown in Table 1.

[0027] Table 1 LMOF dosage optimization Example 3 The ratio of template molecules, functional monomers and cross-linking agents was optimized and analyzed, and the ratio of the fluorescence quenching intensity of PQDs-LMOF@MIP and PQDs-LMOF@NIP by CIP solution (imprinting factor) was used as the measurement index. The specific dosage relationship and imprinting factor are shown in Table 2.

[0028] Table 2 Synthesis ratio optimization Effect Example 1. Study on the properties of PQDs-LMOF@MIP materials In order to have a further understanding of the properties of the prepared materials, the properties of the prepared PQDs-LMOF@MIP material (30 mg of PQDs-LMOF material prepared by LMOF, 0.1mmol: 0.4mmol: 1.6mmol of template molecule, functional monomer and cross-linking agent were studied.

[0029] Figure 1This is a transmission electron microscope image of the PQDs-LMOF@MIP material. As shown in the figure, the surface of PQDs-LMOF is covered with an imprinting layer, and PQDs-LMOF is encapsulated in the imprinting layer, and the imprinting material is successfully prepared.

[0030] Figure 2 This is the X-ray diffraction diagram of PQDs-LMOF@MIP material. As shown in the figure, the characteristic peaks of PQDs at 15.2°, 21.5°, 30.4°, 34.2°, and 37.7° and the diffraction peak of LMOF between 20° and 30° all appear in the PQDs-LMOF curve, proving that the PQDs-LMOF material is composite. After the imprinting layer is further coated in PQDs-LMOF@MIP, the diffraction peak weakens, confirming the successful preparation of PQDs-LMOF@MIP.

[0031] Figure 3 This is a fluorescence selectivity experiment of PQDs-LMOF@MIP material to the same concentration of ENR and its structural analogs ciprofloxacin, ofloxacin, levofloxacin, fleroxacin and pefloxacin. As shown in the figure, the fluorescence responses of PQDs-LMOF@MIP and PQDs-LMOF@NIP to ENR are higher than those of other substances, and the fluorescence response of PQDs-LMOF@MIP to ENR is higher than that of PQDs-LMOF@NIP. The results show that PQDs-LMOF@MIP can specifically recognize ENR and the detection method has good selectivity.

[0032] Figure 4 Figure 2 is the standard curve of fluorescence response of PQDs-LMOF@MIP material to different concentrations of ENR. As shown in the figure, in the range of 0.05-50 μmol / L, the fluorescence response signal F of PQDs-LMOF@MIP and PQDs-LMOF@NIP is LMOF / F PQDs It showed a good linear relationship with ENR concentration; the minimum detection limit of this method was 0.026 μmol / L.

[0033] (II) Application of PQDs-LMOF@MIP materials in ENR analysis and detection The prepared polymer PQDs-LMOF@MIP was added to the sample extract at a solid-liquid ratio of 2 mg: 3 mL, and oscillated at room temperature for 15 min before use for fluorescence spectrophotometer analysis. This method was applied to the determination of ENR in actual samples and compared with the results of HPLC detection. The results are shown in Table 3.

[0034] Table 3 Application of PQDs-LMOF@MIP materials in fluorescence sensing of ENR

Claims

1. A method for preparing an enrofloxacin ratiometric fluorescent probe based on PQDs-LMOF, characterized in that: The following steps are involved: (1) Preparation of LMOF Dissolve europium chloride hexahydrate (EuCl3·6H2O) and succinimide (SI) in appropriate amount of deionized water, stir and ultrasonically treat to obtain Eu 3+ solution and SI solution; then, citric acid was added to Eu 3+ The solution was stirred evenly to obtain a Eu-CA solution; Add the SI solution to the Eu-CA solution and continue stirring, then add the ethanol solution of thenoyl trifluoroacetone and stir for a period of time at an appropriate temperature. Then, adjust the pH value, continue stirring and keep for a period of time, cool to room temperature, centrifuge to separate the precipitate, wash and vacuum dry to obtain LMOF; (2) Preparation of PQDs-LMOF Cesium carbonate (CsCO3), oleic acid and octadecene (ODE) are deoxygenated with N2 and then moved to a heating mantle, and the temperature is raised in stages under a nitrogen atmosphere to carry out a heat preservation reaction to obtain a cesium oleate precursor; Lead bromide and octadecene are evacuated and then connected to nitrogen. After heating to a certain temperature, oleic acid and APTES are added, and the temperature is continued to rise to disperse LMOF in the lead source. Cesium oleate precursor is quickly added, and after a short time, it is cooled in an ice water bath, hydrolyzed, centrifuged, washed, and vacuum dried to obtain PQDs-LMOF. (3) Preparation of PQDs-LMOF@MIP The template molecule was dissolved in n-hexane, functional monomers and PQDs-LMOF were added, a cross-linker was added after prepolymerization and stirred overnight, the product was collected by centrifugation and washed with a mixed solvent of n-hexane and acetic acid, and finally dried under vacuum to obtain PQDs-LMOF@MIP; after removing the template molecule, the above steps were repeated to obtain PQDs-LMOF@NIP.

2. The preparation method according to claim 1, characterized in that: In step (1), the molar ratio of europium chloride hexahydrate to succinimide is 1:3 mmol; the molar ratio of europium chloride hexahydrate to citric acid is 1:0.5; the molar ratio of succinimide to 2-thenoyltrifluoroacetone (TTA) is 3:0.75 mmol, and the mixture is stirred at 50°C for 15 min; the pH is adjusted to 9 using 1 mol / L sodium bicarbonate solution; and the stirring condition is stirring at 50°C for 30 min.

3. The preparation method according to claim 1 or 2, characterized in that: In step (2), the ratio of cesium carbonate (CsCO3), oleic acid and octadecene (ODE) is 0.8 g: 2.5 mL: 30 mL; the ratio of lead bromide, octadecene (ODE), oleic acid and APTES is 0.138 g: 10 mL: 50 μL: 1 mL; the mass ratio of LMOF and lead bromide is 80: 0.13-0.

14.

4. The preparation method according to claim 1 or 3, characterized in that: In step (2), the stepwise heating is firstly heating to 120°C and maintaining for 1 hour; then heating to 140°C and reacting for 2 hours.

5. The preparation method according to any one of claims 1 to 4, characterized in that: In step (2), after adding oleic acid and APTES, the reaction temperature is raised to 120°C at a rotation speed of 500 r / min and maintained for 1 hour; then the temperature is raised to 140°C; the hydrolysis is carried out under stirring in an oil bath at 25°C for 3 hours; 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 molar ratio of the template molecule, the functional monomer and the cross-linking agent is 1:3-5:8-32; the ratio of the template molecule to PQDs-LMOF is 1 mmol:200-1000 mg; and the eluent is composed of n-hexane and acetic acid in a volume ratio of 8:

2.

7. The preparation method according to claim 1 or 6, characterized in that: The template molecule is ENR; the functional monomer is APTES; the cross-linking agent is TMOS; and the vacuum drying temperature is 60°C.

8. Use of an enrofloxacin ratiometric fluorescent probe prepared by the preparation method according to any one of claims 1 to 7 in analyzing and detecting ENR, characterized in that: The following steps are involved: (1) Add the prepared polymer PQDs-LMOF@MIP into the sample extract according to the material-liquid ratio, shake at room temperature, and use for analysis and detection. (2) The sample to be tested is detected and analyzed using a fluorescence spectrophotometer.

9. The use according to claim 8, characterized in that: The ratio of the PQDs-LMOF@MIP to the sample extract was 2 mg:3 mL; and the room temperature shaking time was 15 min.

10. The use according to claim 8 or 9, characterized in that: The conditions for the fluorescence spectrophotometer detection and analysis are: an excitation wavelength of 365 nm, an emission wavelength of 400-700 nm, and a slit width of 5 nm.

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