A ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone and a preparation method thereof

Abstract

The application discloses a ratio type molecular imprinting fluorescent sensor for detecting dexamethasone and a preparation method thereof. The method comprises the following steps: 1, weighing hydrophilic SiO2 nano microspheres and placing the SiO2 nano microspheres in a round-bottom flask, adding γ-methacryloxypropyl trimethoxysilane, triethylamine and toluene, reacting at 110-130 DEG C under N2 atmosphere for 18-24 h, collecting SiO2 microspheres, washing and drying, and obtaining surface-modified SiO2 spherical particles; 2, mixing a danfosyl chloride aqueous solution and a dexamethasone aqueous solution, standing at 30-45 DEG C for 5-15 min, and obtaining a dexamethasone derivative solution; 3, placing the surface-modified SiO2 spherical particles into a round-bottom flask, adding the dexamethasone derivative solution, 4-vinylphenylboronic acid and anhydrous ethanol, standing at 0-4 DEG C for 4-8 h, adding divinylbenzene, dithiothreitol and Eu-TFPA-Phen, reacting at 40-60 DEG C under N2 atmosphere for 6-10 h, centrifuging, washing and drying, and obtaining the ratio type molecular imprinting fluorescent sensor, which can detect dexamethasone in real time, accurately and efficiently.

Description

Technical Field

[0001] This invention belongs to the field of hormone drug detection technology, specifically a ratiometric molecularly imprinted fluorescence sensor for detecting dexamethasone and its preparation method. Background Technology

[0002] Dexamethasone is a synthetic glucocorticoid with anti-inflammatory, anti-allergic, and anti-shock pharmacological effects. my country's food safety laws clearly stipulate that the illegal use of this drug in livestock and poultry farming is strictly prohibited. However, farmers may use this drug illegally to promote animal growth due to the lure of economic benefits. Long-term use of this type of drug will leave residues in the animal's body and further transfer them to animal products. Long-term or excessive intake by humans can disrupt the endocrine system, inhibit adrenal cortex function, and may also cause problems such as osteoporosis, high blood sugar, and decreased immune function. The health hazards are more significant for special groups such as pregnant women and children.

[0003] Currently, the detection of dexamethasone in food safety testing often relies on laboratory methods, specifically chromatography, namely liquid chromatography or liquid chromatography-mass spectrometry. The detection process has the following problems: complex and cumbersome sample pretreatment, large amount of organic reagents used, low detection efficiency, expensive equipment, the need for professional technicians, and limited testing sites.

[0004] Molecularly imprinted sensors offer significant advantages in drug detection, including high specificity, high sensitivity, good stability, low detection cost, and ease of operation. Their core is a recognition element based on molecularly imprinted polymers, capable of accurately identifying target drug molecules and effectively avoiding interference from other components in complex sample matrices. This enables rapid detection of trace drug residues. Furthermore, these sensors are resistant to environmental changes such as acidity, alkali, and temperature, have high reusability, and require no expensive large-scale instruments or complex pretreatment processes. They are suitable for rapid drug screening and quantitative analysis in various scenarios, including food, pharmaceuticals, and the environment.

[0005] Therefore, it is hoped that a molecularly imprinted sensor capable of efficiently detecting dexamethasone can be provided. Summary of the Invention

[0006] The purpose of this invention is to provide a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone and its preparation method, which has the advantages of strong environmental tolerance and high detection efficiency, and can detect dexamethasone in real time, accurately and efficiently.

[0007] This invention is achieved through the following technical solution: A method for preparing a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone includes the following steps: Step 1: Weigh 100-300 mg of hydrophilic SiO2 nanospheres and place them in a round-bottom flask. Then add 5-9 mL of γ-methacryloxypropyltrimethoxysilane and 1-3 mL of triethylamine, followed by 80-100 mL of toluene that has been dehydrated by molecular sieve. After the γ-methacryloxypropyltrimethoxysilane and triethylamine have fully dissolved, introduce N2 into the round-bottom flask to remove oxygen. Place the flask in an oil bath at 110-130°C and stir for 18-24 h. Collect the SiO2 nanospheres, wash and vacuum dry them sequentially to obtain surface-modified SiO2 spherical particles. Step 2: Mix 3-8 mL of 3 mmol / mL dansyl chloride aqueous solution and 5-10 mL of 1 mmol / mL dexamethasone aqueous solution, and let stand at 30-45℃ for 5-15 min to derivatize and obtain a dexamethasone derivative solution with green fluorescence. Step 3: Place 30-50 mg of surface-modified SiO2 spherical particles into a round-bottom flask, add 10-20 mL of dexamethasone derivative solution, then add 3-5 mg of 4-vinylphenylboronic acid and 50-100 mL of anhydrous ethanol. Let stand at 0-4℃ for 4-8 h, then add 10-20 mg of divinylbenzene, 20-50 mg of dithiothreitol and 5-20 mg of red fluorescent Eu-TFPA-Phen powder. Purge the round-bottom flask with N2 to remove oxygen, and react at 40-60℃ for 6-10 h. After the reaction is complete, centrifuge to separate the precipitate, then wash and dry it sequentially to obtain a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone.

[0008] Furthermore, the hydrophilic SiO2 nanospheres of step 1 have a particle size of 20~50 nm.

[0009] Furthermore, the washing in step 1 involves repeatedly washing with methanol and ultrapure water 5 to 6 times.

[0010] Furthermore, the vacuum drying in step 1 is performed using a vacuum drying oven at 80~100℃ for 12~24 h.

[0011] Further, the red fluorescent Eu-TFPA-Phen powder in step 3 is prepared by the following method: 0.1~0.5 mmol Eu(NO3)3·6H2O, 0.15~0.45 mmol o-phenanthroline and 0.15~0.45 mmol 3,4,5,6-tetrafluorophthalic acid are dispersed in 5~10 mL of a mixed solvent of DMF and distilled water in a volume ratio of 1:1. The mixture is ultrasonically treated for 15~30 min, then transferred to a stainless steel container in a high-pressure reactor, sealed and placed in a vacuum drying oven. The mixture is heated at 110~13℃ for 50~72 h, then cooled to 20~30℃ at a cooling rate of 2~5℃ / h, filtered to obtain colorless blocky crystals, washed three times with distilled water, air-dried, and ground into powder to obtain red fluorescent Eu-TFPA-Phen powder.

[0012] Furthermore, the centrifugation in step 3 is performed at a speed of 6000~10000 rpm for 5~10 min.

[0013] Furthermore, the washing in step 3 is performed using anhydrous ethanol until the precipitate shows no blue-green fluorescence under ultraviolet light.

[0014] Furthermore, the drying in step 3 is carried out at 25~35℃.

[0015] A ratiometric molecularly imprinted fluorescence sensor for detecting dexamethasone.

[0016] The present invention has the following beneficial technical effects: 1) First, dexamethasone is derivatized using dansyl chloride to convert it into a dexamethasone derivative with green fluorescence. Then, using the dexamethasone derivative as a template molecule, 4-vinylphenylboronic acid as a functional monomer for molecular imprinting, divinylbenzene and dithiothreitol as crosslinking agents, and red fluorescent Eu-TFPA-Phen powder as a reference fluorescence, a molecularly imprinted polymer is formed and attached to the surface of surface-modified SiO2 spherical particles. The template molecule is then eluted by washing. During this process, the surface-modified SiO2 spherical particles serve as a support, which can confine the imprinting sites to the surface of the support rather than inside the molecularly imprinted polymer, effectively improving the elution efficiency and imprinting effect. The resulting ratiometric molecularly imprinted fluorescent sensor has a specific recognition imprint cavity for dexamethasone, enabling efficient, real-time, and accurate detection of dexamethasone in the environment, food, or pharmaceuticals.

[0017] 2) The molecularly imprinted fluorescent sensor prepared by this invention has the advantages of low cost and strong environmental tolerance. With the help of fluorescence detection, the content of dexamethasone can be estimated visually. It can be used for rapid screening and quantitative analysis of dexamethasone in food, medicine and environment. It does not require large equipment and has good application potential for on-site real-time detection. Attached Figure Description

[0018] Figure 1 The fluorescence response spectra of the ratiometric molecularly imprinted fluorescent sensor prepared in Example 1 of the present invention to dexamethasone solutions of different concentrations are shown. Figure 2 The fluorescence response fitting curves of the ratiometric molecularly imprinted fluorescence sensor prepared in Example 1 of the present invention to dexamethasone solutions of different concentrations are shown. Figure 3 The fluorescence response results of the ratiometric molecularly imprinted fluorescence sensor prepared in Example 1 of this invention to different hormone drugs derived from dansyl chloride are shown. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.

[0020] Example 1 Step 1: Weigh 100 mg of hydrophilic SiO2 nanospheres with a particle size of 20 nm and place them in a round-bottom flask. Then add 5 mL of γ-methacryloxypropyltrimethoxysilane and 1 mL of triethylamine, followed by 80 mL of toluene that has been dehydrated by molecular sieve. After the γ-methacryloxypropyltrimethoxysilane and triethylamine have fully dissolved, introduce N2 into the round-bottom flask to remove oxygen. Place the flask in an oil bath at 110 °C and stir for 18 h. Collect the SiO2 nanospheres and wash them repeatedly with methanol and ultrapure water five times. Transfer them to a vacuum drying oven and dry them under vacuum at 80 °C for 24 h to obtain surface-modified SiO2 spherical particles. Step 2: Mix 3 mL of 3 mmol / mL dansyl chloride aqueous solution and 5 mL of 1 mmol / mL dexamethasone aqueous solution, and let stand at 30℃ for 5 min to derivatize, to obtain a dexamethasone derivative solution with green fluorescence. Step 3: Disperse 0.1 mmol Eu(NO3)3·6H2O, 0.15 mmol o-phenanthroline (Phen) and 0.15 mmol 3,4,5,6-tetrafluorophthalic acid (H2TFPA) in 5 mL of a mixed solvent of DMF and distilled water in a volume ratio of 1:1. Sonicate for 30 min, then transfer to a stainless steel container in a high-pressure reactor, seal and place in a vacuum drying oven. Heat at 110℃ for 72 h, then cool to 30℃ at a cooling rate of 2℃ / h. Filter to obtain colorless blocky crystals, wash three times with distilled water, air dry, and grind into powder to obtain red fluorescent Eu-TFPA-Phen powder. Step 4: Place 30 mg of surface-modified SiO2 spherical particles into a round-bottom flask, add 10 mL of dexamethasone derivative solution, then add 3 mg of 4-vinylphenylboronic acid and 50 mL of anhydrous ethanol. Let stand at 0 °C for 4 h, then add 10 mg of divinylbenzene, 20 mg of dithiothreitol and 5 mg of red fluorescent Eu-TFPA-Phen powder. Purge the round-bottom flask with N2 to remove oxygen, and react at 40 °C for 10 h. After the reaction is complete, centrifuge at 6000 rpm for 10 min to separate the precipitate. Wash with 100 mL of anhydrous ethanol until the precipitate shows no blue-green fluorescence under UV light. Dry at 25 °C to obtain a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone.

[0021] Example 2 Step 1: Weigh 200 mg of hydrophilic SiO2 nanospheres with a particle size of 30 nm and place them in a round-bottom flask. Then add 7 mL of γ-methacryloxypropyltrimethoxysilane and 2 mL of triethylamine, followed by 90 mL of toluene that has been dehydrated by molecular sieve. After the γ-methacryloxypropyltrimethoxysilane and triethylamine have fully dissolved, introduce N2 into the round-bottom flask to remove oxygen. Place the flask in an oil bath at 120 °C and stir for 21 h. Collect the SiO2 nanospheres and wash them repeatedly with methanol and ultrapure water 6 times. Transfer them to a vacuum drying oven and dry them under vacuum at 90 °C for 18 h to obtain surface-modified SiO2 spherical particles. Step 2: Mix 5 mL of 3 mmol / mL dansyl chloride aqueous solution and 8 mL of 1 mmol / mL dexamethasone aqueous solution, and let stand at 40℃ for 10 min to derivatize, to obtain a dexamethasone derivative solution with green fluorescence. Step 3: Disperse 0.2 mmol Eu(NO3)3·6H2O, 0.2 mmol o-phenanthroline (Phen) and 0.2 mmol 3,4,5,6-tetrafluorophthalic acid (H2TFPA) in 6 mL of a mixed solvent of DMF and distilled water in a volume ratio of 1:1. Sonicate for 15 min, then transfer to a stainless steel container in a high-pressure reactor, seal and place in a vacuum drying oven. Heat at 120℃ for 55 h, then cool to 30℃ at a cooling rate of 3℃ / h. Filter to obtain colorless blocky crystals, wash three times with distilled water, air dry, and grind into powder to obtain red fluorescent Eu-TFPA-Phen powder. Step 4: Place 40 mg of surface-modified SiO2 spherical particles into a round-bottom flask, add 15 mL of dexamethasone derivative solution, then add 4 mg of 4-vinylphenylboronic acid and 70 mL of anhydrous ethanol. Let stand at 2°C for 6 h, then add 15 mg of divinylbenzene, 30 mg of dithiothreitol and 15 mg of red fluorescent Eu-TFPA-Phen powder. Purge the round-bottom flask with N2 to remove oxygen, and react at 50°C for 8 h. After the reaction is complete, centrifuge at 8000 rpm for 8 min to separate the precipitate. Wash with 150 mL of anhydrous ethanol until the precipitate shows no blue-green fluorescence under UV light. Dry at 30°C to obtain a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone.

[0022] Example 3 Step 1: Weigh 300 mg of hydrophilic SiO2 nanospheres with a particle size of 50 nm and place them in a round-bottom flask. Then add 9 mL of γ-methacryloxypropyltrimethoxysilane and 3 mL of triethylamine, followed by 100 mL of toluene that has been dehydrated by molecular sieve. After the γ-methacryloxypropyltrimethoxysilane and triethylamine have fully dissolved, introduce N2 into the round-bottom flask to remove oxygen. Place the flask in an oil bath at 130 °C and stir for 18 h. Collect the SiO2 nanospheres and wash them repeatedly with methanol and ultrapure water five times. Transfer them to a vacuum drying oven and dry them under vacuum at 100 °C for 12 h to obtain surface-modified SiO2 spherical particles. Step 2: Mix 8 mL of 3 mmol / mL dansyl chloride aqueous solution and 10 mL of 1 mmol / mL dexamethasone aqueous solution, and let stand at 45℃ for 15 min to derivatize, to obtain a dexamethasone derivative solution with green fluorescence. Step 3: Disperse 0.3 mmol Eu(NO3)3·6H2O, 0.3 mmol o-phenanthroline (Phen) and 0.3 mmol 3,4,5,6-tetrafluorophthalic acid (H2TFPA) in 7 mL of a mixed solvent of DMF and distilled water in a volume ratio of 1:1. Sonicate for 20 min, then transfer to a stainless steel container in a high-pressure reactor, seal and place in a vacuum drying oven. Heat at 130℃ for 50 h, then cool to 30℃ at a cooling rate of 4℃ / h. Filter to obtain colorless blocky crystals, wash three times with distilled water, air dry, and grind into powder to obtain red fluorescent Eu-TFPA-Phen powder. Step 4: Place 50 mg of surface-modified SiO2 spherical particles into a round-bottom flask, add 20 mL of dexamethasone derivative solution, then add 5 mg of 4-vinylphenylboronic acid and 100 mL of anhydrous ethanol. Let stand at 4 °C for 8 h, then add 20 mg of divinylbenzene, 50 mg of dithiothreitol and 20 mg of red fluorescent Eu-TFPA-Phen powder. Purge the round-bottom flask with N2 to remove oxygen, and react at 60 °C for 6 h. After the reaction is complete, centrifuge at 10000 rpm for 5 min to separate the precipitate. Wash with 200 mL of anhydrous ethanol until the precipitate shows no blue-green fluorescence under UV light. Dry at 35 °C to obtain a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone.

[0023] Example 4 Step 1: Weigh 150 mg of hydrophilic SiO2 nanospheres with a particle size of 35 nm and place them in a round-bottom flask. Then add 6 mL of γ-methacryloxypropyltrimethoxysilane and 1.5 mL of triethylamine, followed by 85 mL of toluene that has been dehydrated by molecular sieve. After the γ-methacryloxypropyltrimethoxysilane and triethylamine have fully dissolved, introduce N2 into the round-bottom flask to remove oxygen. Place the flask in an oil bath at 110 °C and stir for 24 h. Collect the SiO2 nanospheres and wash them repeatedly with methanol and ultrapure water five times. Transfer them to a vacuum drying oven and dry them under vacuum at 850 °C for 20 h to obtain surface-modified SiO2 spherical particles. Step 2: Mix 6 mL of 3 mmol / mL dansyl chloride aqueous solution and 6 mL of 1 mmol / mL dexamethasone aqueous solution, and let stand at 35℃ for 12 min to derivatize, to obtain a dexamethasone derivative solution with green fluorescence. Step 3: Disperse 0.4 mmol Eu(NO3)3·6H2O, 0.35 mmol o-phenanthroline (Phen) and 0.35 mmol 3,4,5,6-tetrafluorophthalic acid (H2TFPA) in 8 mL of a mixed solvent of DMF and distilled water in a volume ratio of 1:1. Sonicate for 25 min, then transfer to a stainless steel container in a high-pressure reactor, seal and place in a vacuum drying oven. Heat at 125℃ for 60 h, then cool to 20℃ at a rate of 5℃ / h. Filter to obtain colorless blocky crystals, wash three times with distilled water, air dry, and grind into powder to obtain red fluorescent Eu-TFPA-Phen powder. Step 4: Place 35 mg of surface-modified SiO2 spherical particles into a round-bottom flask, add 10 mL of dexamethasone derivative solution, then add 3 mg of 4-vinylphenylboronic acid and 80 mL of anhydrous ethanol. Let stand at 1°C for 5 h, then add 12 mg of divinylbenzene, 35 mg of dithiothreitol and 10 mg of red fluorescent Eu-TFPA-Phen powder. Purge the round-bottom flask with N2 to remove oxygen, and react at 45°C for 9 h. After the reaction is complete, centrifuge at 7000 rpm for 9 min to separate the precipitate. Wash with 150 mL of anhydrous ethanol until the precipitate shows no blue-green fluorescence under UV light. Dry at 25°C to obtain a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone.

[0024] Example 5 Step 1: Weigh 250 mg of hydrophilic SiO2 nanospheres with a particle size of 40 nm and place them in a round-bottom flask. Then add 8 mL of γ-methacryloxypropyltrimethoxysilane and 2.5 mL of triethylamine, followed by 95 mL of toluene that has been dehydrated by molecular sieve. After the γ-methacryloxypropyltrimethoxysilane and triethylamine have fully dissolved, introduce N2 into the round-bottom flask to remove oxygen. Place the flask in an oil bath at 125 °C and stir for 20 h. Collect the SiO2 nanospheres and wash them repeatedly with methanol and ultrapure water 6 times. Transfer them to a vacuum drying oven and dry them under vacuum at 95 °C for 15 h to obtain surface-modified SiO2 spherical particles. Step 2: Mix 7 mL of 3 mmol / mL dansyl chloride aqueous solution and 9 mL of 1 mmol / mL dexamethasone aqueous solution, and let stand at 45℃ for 8 min to derivatize, to obtain a dexamethasone derivative solution with green fluorescence. Step 3: Disperse 0.5 mmol Eu(NO3)3·6H2O, 0.45 mmol o-phenanthroline (Phen) and 0.45 mmol 3,4,5,6-tetrafluorophthalic acid (H2TFPA) in 10 mL of a mixed solvent of DMF and distilled water in a volume ratio of 1:1. Sonicate for 30 min, then transfer to a stainless steel container in a high-pressure reactor, seal and place in a vacuum drying oven. Heat at 115℃ for 65 h, then cool to 25℃ at a cooling rate of 2℃ / h. Filter to obtain colorless blocky crystals, wash three times with distilled water, air dry, and grind into powder to obtain red fluorescent Eu-TFPA-Phen powder. Step 4: Place 45 mg of surface-modified SiO2 spherical particles into a round-bottom flask, add 20 mL of dexamethasone derivative solution, then add 4 mg of 4-vinylphenylboronic acid and 90 mL of anhydrous ethanol. Let stand at 3°C ​​for 7 h, then add 18 mg of divinylbenzene, 45 mg of dithiothreitol and 13 mg of red fluorescent Eu-TFPA-Phen powder. Purge the round-bottom flask with N2 to remove oxygen, and react at 55°C for 7 h. After the reaction is complete, centrifuge at 9000 rpm for 6 min to separate the precipitate. Wash with 200 mL of anhydrous ethanol until the precipitate shows no blue-green fluorescence under UV light. Dry at 35°C to obtain a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone.

[0025] The ratiometric molecularly imprinted fluorescent sensors prepared in Examples 1 to 5 all exhibit uniform nanospheres. Under 365 nm ultraviolet light excitation, they show red fluorescence. When used to detect dexamethasone, they are first derivatized with dansyl chloride to obtain dansyl chloride-derived dexamethasone. Then, the fluorescent sensor is used to identify the dansyl chloride-derived dexamethasone. Under ultraviolet light excitation, the green fluorescence of the fluorescent sensor increases with the increase of dexamethasone concentration, while the red fluorescence intensity remains unchanged. A linear functional relationship between the ratio of green fluorescence to red fluorescence and the dexamethasone content is established, thereby realizing the detection of dexamethasone.

[0026] To verify the performance of the ratiometric molecularly imprinted fluorescent sensors prepared in Examples 1 to 5, the ratiometric molecularly imprinted fluorescent sensor prepared in Example 1 was used as a representative, and its various performance characteristics were tested. The test process and results are as follows: 1) Fluorescence detection performance of ratiometric molecularly imprinted fluorescence sensors Add a 1 mg / L dexamethasone standard solution to a 3 mmol / mL dansyl chloride aqueous solution to prepare test stock solutions of different concentrations. Incubate for 15 min to derivatize and allow dexamethasone to react with dansyl chloride. Simultaneously, disperse the ratiometric molecularly imprinted fluorescent sensor prepared in Example 1 in ultrapure water to prepare a 5.0 mg / mL fluorescent sensor solution, and divide it evenly into several 5 mL portions. Add 10 μL of the test stock solution to each portion of the fluorescent sensor solution to make the concentrations of the test stock solution in each portion of the fluorescent sensor solution 0.25 μg / L, 0.5 μg / L, 1.0 μg / L, 5.0 μg / L, 10 μg / L, 20 μg / L, 40 μg / L, 60 μg / L, 80 μg / L, 100 μg / L, 125 μg / L, 150 μg / L, 175 μg / L, 200 μg / L, etc. Dexamethasone solutions of 250 μg / L, 300 μg / L, and 400 μg / L were incubated at room temperature for 10 min. A fluorescence sensor solution without the added test stock solution served as the control group (i.e., the concentration of the test stock solution in the fluorescence sensor solution was 0 μg / L). The fluorescence intensity of each dexamethasone solution and the control group was measured using a fluorescence spectrometer. The results are as follows: Figure 1 As shown, and with fluorescence intensity ratio I 443 / I 580 The values ​​are plotted on the ordinate and the concentration of the mother liquor being tested is plotted on the abscissa as follows: Figure 2 The fitted curve shown is given, where: by Figure 1 It can be seen that as the concentration of dexamethasone solution increases, the fluorescence intensity at 443 nm gradually increases, while the reference fluorescence intensity at 580 nm remains almost unchanged; Figure 2 It can be seen that I 443 / I 580 The value showed a linear fit with the change in dexamethasone solution concentration, and the fitting curve R... 2 The value is greater than 0.997; it can be seen that the fluorescence sensor prepared in this invention has a good linear relationship with the response of dexamethasone derived from dansyl chloride, and can realize the quantitative detection of dexamethasone. The detection limit of dexamethasone is calculated to be 1.5 ng / mL using 3σ / k, where σ is the standard deviation of the control group and k is the slope of the calibration curve.

[0027] 2) Selectivity of ratiometric molecularly imprinted fluorescence sensors for different hormone drugs The ratiometric molecularly imprinted fluorescent sensor prepared in Example 1 was dispersed in ultrapure water to prepare a 5.0 mg / mL fluorescent sensor solution, which was then divided into several aliquots of 5 mL each. The same concentration of different hormone drugs derived from dansyl chloride was added to each aliquot. The solutions were incubated at room temperature for 10 min. After fluorescence stabilization, the fluorescence spectrum of each aliquot was measured. The selectivity of the sensor was evaluated by the ratio of fluorescence intensity at 443 nm to 580 nm. The results are as follows: Figure 3 As shown, it can be seen that dexamethasone derived from dansyl chloride alone produced a significant fluorescence response at 443 nm. 443 / I 580 The value is significantly higher than that of the test groups corresponding to other hormone drugs, indicating that the fluorescence sensor prepared in Example 1 has no obvious fluorescence response to other hormone drugs, but has a good selective response to dexamethasone.

[0028] 3) Actual sample detection performance of the ratiometric molecularly imprinted fluorescence sensor The ratiometric molecularly imprinted fluorescent sensor prepared in Example 1 was dispersed in ultrapure water to prepare a fluorescent sensor solution with a concentration of 5.0 mg / mL. This solution was divided into four 5 mL portions for later use. 20 g of milk powder sample was weighed and divided into four equal portions. Each portion was placed in a 50 mL centrifuge tube, and 20 mL of 50% ethanol aqueous solution was added and thoroughly mixed. The mixture was centrifuged at 5000 rpm for 5 min. 2 mL of the supernatant was added to 10 μL of 3 mmol / mL dansyl chloride aqueous solution, and incubated at room temperature for 10 min to obtain four samples. One of these samples was added to one of the pre-prepared sensor solutions. After the fluorescence stabilized, the fluorescence spectrum of each solution was measured. Figure 2 The dexamethasone content in each fluorescence sensor solution was calculated using the fitted curve. The remaining three samples were then spiked for recovery experiments at concentrations of 10 μg / kg, 20 μg / kg, and 30 μg / kg, respectively. These spikes were then added to the remaining three fluorescence sensor solutions accordingly. Figure 2 The dexamethasone content in each fluorescent sensor solution was calculated using the fitted curve, and the recovery rate was also calculated. Simultaneously, the dexamethasone content in milk powder samples was determined as a control according to the method specified in GB / T 22978-2008. The results are shown in Table 1. Table 1. Measured values ​​of dexamethasone and results of spiked experiments in milk powder samples. As can be seen from Table 1, the dexamethasone content in the milk powder sample determined by the ratiometric fluorescence sensor prepared in Example 1 (i.e., fluorescence detection) is similar to the result determined by liquid chromatography-tandem mass spectrometry (i.e., LC-MS) specified in GB / T 22978-2008; moreover, the spiked recovery experiment results show that the recovery rate is 95%~102%, indicating that the ratiometric fluorescence sensor prepared in this invention has good reliability when used to detect dexamethasone in food.

Claims

1. A method for preparing a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone, characterized in that, Includes the following steps: Step 1: Weigh 100-300 mg of hydrophilic SiO2 nanospheres and place them in a round-bottom flask. Then add 5-9 mL of γ-methacryloxypropyltrimethoxysilane and 1-3 mL of triethylamine, followed by 80-100 mL of toluene that has been dehydrated by molecular sieve. After the γ-methacryloxypropyltrimethoxysilane and triethylamine have fully dissolved, introduce N2 into the round-bottom flask to remove oxygen. Place the flask in an oil bath at 110-130°C and stir for 18-24 h. Collect the SiO2 nanospheres, wash and vacuum dry them sequentially to obtain surface-modified SiO2 spherical particles. Step 2: Mix 3-8 mL of 3 mmol / mL dansyl chloride aqueous solution and 5-10 mL of 1 mmol / mL dexamethasone aqueous solution, and let stand at 30-45℃ for 5-15 min to derivatize and obtain a dexamethasone derivative solution with green fluorescence. Step 3: Place 30-50 mg of surface-modified SiO2 spherical particles into a round-bottom flask, add 10-20 mL of dexamethasone derivative solution, then add 3-5 mg of 4-vinylphenylboronic acid and 50-100 mL of anhydrous ethanol. Let stand at 0-4℃ for 4-8 h, then add 10-20 mg of divinylbenzene, 20-50 mg of dithiothreitol and 5-20 mg of red fluorescent Eu-TFPA-Phen powder. Purge the round-bottom flask with N2 to remove oxygen, and react at 40-60℃ for 6-10 h. After the reaction is complete, centrifuge to separate the precipitate, then wash and dry it sequentially to obtain a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone.

2. The method for preparing a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone according to claim 1, characterized in that, The hydrophilic SiO2 nanospheres obtained in step 1 have a particle size of 20~50 nm.

3. The method for preparing a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone according to claim 1, characterized in that, The washing process in step 1 involves repeatedly washing with methanol and ultrapure water 5 to 6 times.

4. The method for preparing a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone according to claim 1, characterized in that, The vacuum drying in step 1 is carried out in a vacuum drying oven at 80~100℃ for 12~24 hours.

5. The method for preparing a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone according to claim 1, characterized in that, The red fluorescent Eu-TFPA-Phen powder in step 3 was prepared by the following method: 0.1-0.5 mmol Eu(NO3)3·6H2O, 0.15-0.45 mmol o-phenanthroline, and 0.15-0.45 mmol 3,4,5,6-tetrafluorophthalic acid were dispersed in 5-10 mL of a mixed solvent consisting of DMF and distilled water in a 1:1 volume ratio. The mixture was ultrasonically treated for 15-30 min, then transferred to a stainless steel container in a high-pressure reactor, sealed, and placed in a vacuum drying oven. The mixture was heated at 110-13℃ for 50-72 h, then cooled to 20-30℃ at a rate of 2-5℃ / h. After filtration, colorless blocky crystals were obtained, washed three times with distilled water, air-dried, and ground into powder to obtain the red fluorescent Eu-TFPA-Phen powder.

6. The method for preparing a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone according to claim 1, characterized in that, The centrifugation in step 3 is performed at a speed of 6000~10000 rpm for 5~10 minutes.

7. The method for preparing a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone according to claim 1, characterized in that, The washing in step 3 involves washing with anhydrous ethanol until the precipitate shows no blue-green fluorescence under ultraviolet light.

8. The method for preparing a ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone according to claim 1, characterized in that, The drying in step 3 is carried out at 25~35℃.

9. A ratiometric molecularly imprinted fluorescent sensor for detecting dexamethasone prepared by the method according to any one of claims 1 to 8.

Images