Dual-emission molecular imprinting ratiometric fluorescence sensor, preparation method thereof and application of dual-emission molecular imprinting ratiometric fluorescence sensor in aureomycin detection

Abstract

The invention discloses a dual-emission molecular imprinting ratiometric fluorescence sensor, a preparation method thereof and application of the dual-emission molecular imprinting ratiometric fluorescence sensor in aureomycin detection, and belongs to the technical field of chemical sensors. The preparation method of the sensor comprises the following steps: preparing silicon dioxide-quantum dot composite nanoparticles, and preparing a molecularly imprinted polymer by taking salicylamide as a template molecule; and mixing the molecularly imprinted polymer and the silicon dioxide-quantum dot composite nanoparticles in an HEPES buffer solution by adopting a post-mixing strategy to construct the dual-emission molecularly imprinted ratiometric fluorescence sensor. The double-emission molecular imprinting ratiometric fluorescence sensor has the beneficial effects that the double-emission molecular imprinting ratiometric fluorescence sensor provided by the invention is rich in fluorescence color change, rapid, high-sensitivity and visual detection of aureomycin can be realized, the whole detection process is simple, convenient and rapid, and the double-emission molecular imprinting ratiometric fluorescence sensor is suitable for being widely popularized in practical application.

Description

Technical Field

[0001] This invention relates to a fluorescence sensor, its preparation method, and its application, specifically to a dual-emission molecularly imprinted ratio fluorescence sensor, its preparation method, and its application in the detection of chlortetracycline, belonging to the field of chemical sensor technology. Background Technology

[0002] Chlortetracycline (CTC) is a representative tetracycline antibiotic produced by *Streptomyces aureus*, possessing bactericidal and sterilizing effects. It is widely used for the prevention and treatment of diseases caused by bacterial infections and can also be used as a feed additive to promote animal growth. However, chlortetracycline has a relatively long half-life in animal tissues, and uncontrolled use can lead to excessive residues in livestock and aquaculture products, seriously affecting product quality and safety. Furthermore, it accumulates in the human body through the food chain, posing a significant threat to human health, such as aplastic anemia and myeloid leukemia. In addition, chlortetracycline residues and its transformation products in environmental water bodies not only inhibit microbial growth and metabolism but also increase bacterial resistance and exacerbate the spread of antibiotic resistance genes, posing a threat to public health. Therefore, there is an urgent need for a sensitive and selective detection method to detect chlortetracycline residues in the environment and food.

[0003] Currently, various analytical methods have been developed for the detection of chlortetracycline, including chromatographic methods such as high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), as well as other methods such as enzyme-linked immunosorbent assay (ELISA) and biological methods. Although these methods have high sensitivity, their widespread application is limited by factors such as complex equipment, long detection time, and high requirements for operator expertise.

[0004] Fluorescence analysis methods have seen rapid development in fields such as chemical sensing and bioanalysis due to their advantages of high specificity, high sensitivity, simple operation, and short processing time. Ratio fluorescence methods typically employ two or more independent fluorescence signals, utilizing the relative ratio of intensities between fluorescence emission peaks to detect analytes. Compared to a single fluorescence signal, this method effectively avoids background absorption, reduces errors, and makes color changes more easily identifiable by the naked eye, improving the accuracy of visual detection. In recent years, research has combined ratio fluorescence with molecular imprinting technology to overcome the lack of output signals in molecularly imprinted polymers, constructing highly sensitive and selective molecularly imprinted fluorescence sensors. This improves the sensor's ability to detect antibiotic residues in complex matrices, for example: (1) Sun Dani et al. constructed dual-emission and triple-emission molecular imprint ratio fluorescence sensors by using strategies such as embedding, imprinting and mixing, and imprinting and modification, and realized rapid, accurate and visual detection of ciprofloxacin hydrochloride and enrofloxacin in environmental water. (2) Cao Jinyu et al. used a novel dual-signal ratio fluorescence sensor based on europium coordination polymers doped with carbon quantum dots and combined with molecularly imprinted polymers for the accurate detection of tetracycline and oxytetracycline.

[0005] However, to date, no molecularly imprinted ratio fluorescence sensors specifically designed for the detection of chlortetracycline have been reported. Summary of the Invention

[0006] The purpose of this invention is to combine ratiometric fluorescence with molecular imprinting technology to provide a dual-emission molecular imprinted ratiometric fluorescence sensor with high sensitivity and selectivity for chlortetracycline in complex matrices, and a simple, convenient and rapid preparation method for the dual-emission molecular imprinted ratiometric fluorescence sensor.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for fabricating a dual-emission molecularly imprinted ratiometric fluorescence sensor includes the following steps: (1) Preparation of silica-quantum dot composite nanoparticles: ultrapure water and silica solution were mixed, and then 3-aminopropyltriethoxysilane was added to obtain a mixed solution. After stirring for 30 min, N-acetyl-L-cysteine-modified cadmium telluride quantum dot solution was added and stirred for another 30 min. Then, ammonia monohydrate and tetraethoxysilane were added and stirred in the dark for 12±1 h to obtain silica-quantum dot composite nanoparticles. (2) Preparation of molecularly imprinted polymer: Salicylic amide was dissolved in ethanol, and ultrapure water, silica solution and 3-aminopropyltriethoxysilane were added in sequence to obtain a mixed solution. After stirring for 30 min, hexadecyltrimethylammonium bromide solution and sodium hydroxide solution were added and stirring was continued for 30 min. Then, ammonia monohydrate and tetraethoxysilane were added and stirring was continued for 3 h. Finally, aluminum chloride solution was added and stirring was carried out for 12 ± 1 h to obtain a molecularly imprinted polymer with salicylic amide template molecule. Salicylic amide was eluted to obtain the final molecularly imprinted polymer. (3) Construction of a dual-emission molecularly imprinted ratio fluorescence sensor: The molecularly imprinted polymer prepared in step (2) and the silica-quantum dot composite nanoparticles prepared in step (1) are mixed in HEPES buffer to construct a dual-emission molecularly imprinted ratio fluorescence sensor.

[0008] Preferably, in steps (1) and (2), the method for preparing the silica solution is as follows: Ethanol and ultrapure water were mixed, and ammonia water with a mass concentration of 25%-28% was added. Tetraethoxysilane and ethanol were added dropwise using a constant pressure dropping funnel under the conditions of room temperature and magnetic stirring. The mixture was magnetically stirred for 12 hours at room temperature to obtain silica nanoparticles. After purification, the nanoparticles were dispersed in ultrapure water to obtain a silica solution.

[0009] Preferably, in step (1), the method for preparing the N-acetyl-L-cysteine-modified cadmium telluride quantum dots is as follows: (i) Add ultrapure water, ethanol, sodium borohydride and tellurium powder to the reaction vessel and react for 4 hours under a water bath at 40°C to form a bright purple supernatant containing sodium tellurium hydride. Use this supernatant as a precursor for the synthesis of cadmium telluride quantum dots. (ii) Cadmium chloride and N-acetyl-L-cysteine ​​were dissolved in ultrapure water to obtain a mixed solution. After adjusting the pH to 9, nitrogen gas was purged to remove oxygen. Then, the newly prepared precursor was added to the mixed solution. The mixture was stirred at room temperature for 10 min and then heated under reflux for 1 h to obtain N-acetyl-L-cysteine-modified cadmium telluride quantum dots.

[0010] Preferably, in step (1), the ratio of ultrapure water, silica solution, 3-aminopropyltriethoxysilane, N-acetyl-L-cysteine-modified cadmium telluride quantum dot solution, ammonia monohydrate and tetraethoxysilane is 13 mL: 400 μL: 10 μL: 2 mL: 100 μL: 100 μL.

[0011] Preferably, in step (2), the ratio of salicylamide, ethanol, ultrapure water, silica solution, 3-aminopropyltriethoxysilane, hexadecyltrimethylammonium bromide, sodium hydroxide, ammonia monohydrate, tetraethoxysilane and aluminum chloride is 10 mg: 0.5 mL: 13 mL: 400 μL: 15 μL: 0.1 mmol: 0.05 mmol: 120 μL: 120 μL: 1 mmol.

[0012] Preferably, in step (3), the method for constructing the dual-emission molecularly imprinted ratiometric fluorescence sensor is as follows: (i) Disperse the molecularly imprinted polymer obtained in step (2) into ultrapure water; (ii) Disperse the silica-quantum dot composite nanoparticles prepared in step (1) into an amount of ultrapure water equal to that in step (i); (iii) The molecularly imprinted polymer dispersed in ultrapure water and the silica-quantum dot composite nanoparticles were mixed in HEPES buffer at a volume ratio of 10:1.

[0013] A dual-emission molecularly imprinted ratiometric fluorescence sensor was prepared by the aforementioned method.

[0014] The aforementioned application of the dual-emission molecularly imprinted ratio fluorescence sensor in the detection of chlortetracycline.

[0015] The advantages of this invention are: (1) In this invention, aluminum ions are used as complexing agents and auxiliary functional monomers to participate in the imprinting process. The green fluorescence of chlortetracycline itself is used as the response signal. Chlortetracycline complexes with aluminum ions in the imprinting site. The coordination effect of aluminum ions restricts the rotation of chlortetracycline molecules, and the inherent green fluorescence peak of chlortetracycline is enhanced. Meanwhile, the red fluorescence of quantum dots remains stable as an inert internal standard. This invention creatively constructs a dual-emission molecular imprinting ratio fluorescence sensor, realizing the selective recognition of chlortetracycline.

[0016] (2) The dual-emission molecular imprint ratio fluorescence sensor provided by the present invention has rich fluorescence color changes, realizing rapid, highly sensitive and visual detection of chlortetracycline.

[0017] (3) The detection of chlortetracycline using the dual-emission molecular imprint ratio fluorescence sensor provided by the present invention is simple, convenient and fast, and is suitable for widespread application in practical applications.

[0018] (4) The dual-emission molecular imprinted ratio fluorescence sensor is prepared using the method provided by the present invention. The entire preparation process is simple, convenient and fast. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the fabrication process of a dual-emission molecularly imprinted ratiometric fluorescence sensor; Figure 2 It includes silica nanoparticles (SiO2), silica-quantum dot composite nanoparticles (SiO2-QDs), and molecularly imprinted polymers (Al). 3+ -MIPs) and non-imprinted polymers (Al) 3+ Transmission electron microscopy image of -NIPs; Figure 3 It includes silica nanoparticles (SiO2), silica-quantum dot composite nanoparticles (SiO2-QDs), and molecularly imprinted polymers (Al). 3+ -MIPs) and non-imprinted polymers (Al) 3+ Fourier transform infrared spectra of -NIPs; Figure 4 These are graphs showing the changes in fluorescence emission peak intensity of the dual-emission molecularly imprinted ratio fluorescence sensor as the concentration of chlortetracycline increases, and the color changes of the dual-emission molecularly imprinted ratio fluorescence sensor under a 365nm UV lamp. In particular, A is the graph showing the changes in fluorescence emission peak intensity of the dual-emission molecularly imprinted ratio fluorescence sensor as the concentration of chlortetracycline increases, and B is the graph showing the color changes of the dual-emission molecularly imprinted ratio fluorescence sensor under a 365nm UV lamp as the concentration of chlortetracycline increases. Figure 5The graphs show the changes in fluorescence emission peak intensity of the non-blot fluorescent sensor as the concentration of chlortetracycline increases, and the color changes of the non-blot fluorescent sensor under a 365nm UV lamp. In the graphs, A is the change in fluorescence emission peak intensity of the non-blot fluorescent sensor as the concentration of chlortetracycline increases, and B is the color change of the non-blot fluorescent sensor under a 365nm UV lamp as the concentration of chlortetracycline increases. Figure 6 The graphs show the selective results of the dual-emission molecularly imprinted ratiometric fluorescence sensor and the non-imprinted fluorescence sensor for different antibiotics and antibiotic analogs, as well as the color changes of each sensor under a 365nm UV lamp. In particular, A is the selective experimental results of the dual-emission molecularly imprinted ratiometric fluorescence sensor and the non-imprinted fluorescence sensor for different antibiotics and antibiotic analogs, and B is the color change of each sensor under a 365nm UV lamp. Detailed Implementation

[0020] The dual-emission molecularly imprinted ratiometric fluorescence sensor provided by this invention is composed of a molecularly imprinted polymer (Al). 3+ The nanoparticles were constructed using a post-mixing strategy, consisting of MIPs and silica-quantum dot composite nanoparticles (SiO2-QDs).

[0021] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0022] I. Preparation of silica-quantum dot composite nanoparticles Silica-quantum dot composite nanoparticles were prepared using the sol-gel method with silica nanoparticles as the core support material.

[0023] 1. Preparation of N-acetyl-L-cysteine-modified cadmium telluride quantum dots

[0024] 500 μL of ultrapure water, 1.5 mL of ethanol, 0.04 g of sodium borohydride and 0.038 g of tellurium powder were added to a three-necked flask and reacted in a water bath at 40 °C for 4 h to form a bright purple supernatant containing sodium telluride. This supernatant was used as a precursor for the synthesis of cadmium telluride quantum dots.

[0025] 0.684 g of cadmium chloride and 0.762 g of N-acetyl-L-cysteine ​​were dissolved in 75 mL of ultrapure water to obtain a mixed solution. The pH of the mixed solution was adjusted to 9 using 1 mol / L sodium hydroxide solution. Nitrogen gas was purged into the mixed solution for 30 min to remove oxygen. 1 mL of the newly prepared precursor was added to the mixed solution, and the mixture was stirred at room temperature for 10 min, followed by reflux for 1 h to obtain N-acetyl-L-cysteine-modified cadmium telluride quantum dots, hereinafter referred to as CdTe quantum dots. These CdTe quantum dots fluoresce red under 365 nm ultraviolet light.

[0026] 2. Preparation of silica solution

[0027] 30 mL of ethanol and 50 mL of ultrapure water were mixed, and 10 mL of ammonia solution with a mass concentration of 25%-28% was added. Under constant pressure and magnetic stirring conditions at room temperature, 5 mL of tetraethoxysilane and 20 mL of ethanol were added dropwise using a constant pressure dropping funnel. The mixture was magnetically stirred for 12 hours at room temperature to obtain silica nanoparticles. The silica nanoparticles were then purified by centrifugation and washing, and finally dispersed in 50 mL of ultrapure water to obtain a silica solution for later use.

[0028] 3. Preparation of silica-quantum dot composite nanoparticles

[0029] Reference Figure 1 13 mL of ultrapure water and 400 μL of the previously prepared silica solution were mixed, and then 10 μL of 3-aminopropyltriethoxysilane (APTES) was added to obtain a mixed solution. The mixed solution was stirred for 30 min, and then 2 mL of the previously prepared CdTe quantum dot solution was added, followed by stirring for another 30 min. Then, 100 μL of ammonia monohydrate (NH3·H2O) and 100 μL of tetraethoxysilane (TEOS) were added, and the mixture was stirred in the dark for 12 ± 1 h to obtain silica-quantum dot composite nanoparticles (SiO2-QDs). The obtained SiO2-QDs were washed three times with ultrapure water and then stored in 2 mL of ultrapure water at room temperature in the dark for later use.

[0030] II. Preparation of Molecularly Imprinted Polymers and Non-Imprinted Polymers: Molecularly imprinted polymers and non-imprinted polymers were prepared using the sol-gel method with silica nanoparticles as the core support material.

[0031] Reference Figure 110 mg of salicylamide (similar in structure and properties to chlortetracycline, used as a virtual template, 0.07 mmol) was weighed and dissolved in 0.5 mL of ethanol. Then, 13 mL of ultrapure water, 400 μL of the previously prepared silica solution, and 15 μL of 3-aminopropyltriethoxysilane (APTES) were added sequentially to obtain a mixed solution. The mixture was stirred for 30 min, then 0.5 mL of 0.2 mol / L hexadecyltrimethylammonium bromide (CTAB) solution and 50 μL of 1 mol / L sodium hydroxide solution were added, and stirring continued for another 30 min. Next, 120 μL of ammonia monohydrate (NH3·H2O) and 120 μL of tetraethoxysilane (TEOS) were added, and stirring continued for 3 h. Finally, 1 mL of 1 mol / L aluminum chloride solution (1 mmol) was added, and stirring was continued for 12 ± 1 h to obtain a molecularly imprinted polymer with the template molecule salicylamide. The obtained molecularly imprinted polymer with the template molecule salicylamide was first eluted to remove the salicylamide using a mixture of ethanol and acetonitrile (volume ratio 4:1). Then, the ethanol and acetonitrile were washed away with ultrapure water to obtain the final molecularly imprinted polymer (Al). 3+ -MIPs). This final molecularly imprinted polymer (Al) 3+ Disperse the MIPs in 2 mL of ultrapure water and set aside.

[0032] As a control, the same steps were used but without the addition of salicylamide to prepare a non-imprinted polymer (Al). 3+ -NIPs). This non-imprinted polymer (Al) 3+ Disperse the -NIPs in 2 mL of ultrapure water for later use.

[0033] III. Observation of SiO2, SiO2-QDs, and Al 3+ -MIPs and Al 3+ Microstructure and Fourier transform infrared spectra of -NIPs The previously prepared silica nanoparticles (SiO2), silica-quantum dot composite nanoparticles (SiO2-QDs), and molecularly imprinted polymers (Al) were used. 3+ -MIPs) and non-imprinted polymers (Al) 3+ -NIPs), vacuum dried, their microstructures were observed by transmission electron microscopy, and their Fourier transform infrared spectra were observed by Fourier transform infrared spectroscopy.

[0034] SiO2, SiO2-QDs, Al 3+ -MIPs and Al 3+ The transmission electron microscopy results of -NIPs are shown in [the image]. Figure 2 .

[0035] Depend on Figure 2It can be seen that the morphology of all four nanomaterials is approximately spherical. Compared with the surface of SiO2-QDs, Al... 3+ -MIPs and Al 3+ -NIPs have a rougher surface, Al 3+ - The imprinted shell thickness of MIPs is approximately 5-8 nm.

[0036] SiO2, SiO2-QDs, Al 3+ -MIPs and Al 3+ The Fourier transform infrared spectroscopy results for -NIPs are shown below. Figure 3 .

[0037] Depend on Figure 3 It can be seen that: SiO2, SiO2-QDs, Al 3+ -MIPs and Al 3+ The same vibrational peaks appeared in the Fourier transform infrared spectra of -NIPs, among which, the peak at 801 cm⁻¹ was observed. -1 The characteristic peak at 1080 cm⁻¹ belongs to the tensile vibration of Si-O. -1 and 945cm -1 The strong absorption bands at these locations indicate the presence of tensile vibration bands in Si-O-Si or Si-OC structures, suggesting the presence of SiO2-QDs and Al... 3+ -MIPs and Al 3+ SiO2 was successfully introduced into all NIPs; located at 3430 cm⁻¹ -1 The characteristic peak at this point is attributed to the vibrations of OH and NH, indicating that 3-aminopropyltriethoxysilane successfully participated in the synthesis of SiO2-QDs and Al. 3+ -MIPs and Al 3+ -NIPs; located at 1631cm -1 1552cm -1 The characteristic peaks at the point are attributed to the stretching vibrations of C=O and -NH, indicating that N-acetyl-L-cysteine-modified cadmium telluride quantum dots have been successfully involved in the synthesis of SiO2-QDs.

[0038] IV. Construction of Dual-Emission Molecular Imprinted Ratio-Fluorescence Sensors and Non-Imprinted Fluorescence Sensors

[0039] A post-mixing strategy was adopted to mix the previously prepared molecularly imprinted polymer (Al) 3+ -MIPs) and silica-quantum dot composite nanoparticles (SiO2-QDs) were mixed in HEPES buffer at pH 6.5 and a concentration of 10 mmol / L. 3+ A dual-emission molecularly imprinted ratiometric fluorescence sensor was constructed by using MIPs, SiO2-QDs, and HEPES buffer in a volume ratio of 10:1:88.

[0040] As a control, the previously prepared non-imprinted polymer (Al) was used. 3+ -NIPs) and silica-quantum dot composite nanoparticles (SiO2-QDs) were mixed in HEPES buffer at pH 6.5 and a concentration of 10 mmol / L. 3+ A non-blot fluorescent sensor was constructed by using NIPs, SiO2-QDs, and HEPES buffer in a volume ratio of 10:1:88.

[0041] V. Detection of chlortetracycline using a dual-emission molecularly imprinted ratiometric fluorescence sensor and an unimprinted fluorescence sensor

[0042] Under 365 nm ultraviolet light excitation, chlortetracycline exhibits a green emission peak at 500 nm and a red emission peak at 650 nm for CdTe quantum dots. The fluorescence intensity of the solution was measured using a fluorescence spectrophotometer, and the quantitative detection of chlortetracycline was achieved by utilizing the correlation between the change in the ratio of red to green fluorescence intensity and the concentration of chlortetracycline.

[0043] 1. The effect of chlortetracycline concentration on detection

[0044] Take 100 μL of the molecularly imprinted polymer (Al) prepared earlier. 3+ 10 μL of the previously prepared silica-quantum dot composite nanoparticles (SiO2-QDs) and 10 μL of the pre-prepared silica-quantum dot composite nanoparticles were added to 880 μL of HEPES buffer (10 mmol / L, pH 6.5) to construct a dual-emission molecularly imprinted ratiometric fluorescence sensor.

[0045] As a control, 100 μL of the previously prepared non-imprinted polymer (Al) was taken. 3+ 10 μL of the previously prepared silica-quantum dot composite nanoparticles (SiO2-QDs) and 10 μL of the silica-quantum dot composite nanoparticles (SiO2-QDs) were added to 880 μL of HEPES buffer (10 mmol / L, pH 6.5) to construct a non-blot fluorescent sensor.

[0046] A series of chlortetracycline solutions of different concentrations were prepared using ultrapure water. The concentrations of chlortetracycline were 0.05 μmol / L, 0.15 μmol / L, 0.5 μmol / L, 1.0 μmol / L, 2.5 μmol / L, 5.0 μmol / L, 10.0 μmol / L, 15.0 μmol / L, 18.75 μmol / L, and 25.0 μmol / L.

[0047] Take 10 μL of chlortetracycline solution of different concentrations and add it to the above dual-emission molecularly imprinted ratiometric fluorescence sensor (990 μL) or non-imprinted fluorescence sensor (990 μL). After mixing and shaking, measure the fluorescence intensity of each sample using a fluorescence spectrophotometer.

[0048] The fluorescence emission peak intensity change of the dual-emission molecularly imprinted ratiometric fluorescence sensor with increasing chlortetracycline concentration and its color change under a 365nm UV lamp are shown in the figure. Figure 4 .

[0049] Depend on Figure 4 It can be observed that as the concentration of chlortetracycline increases, the intensity of the emission peak (650 nm) of the CdTe quantum dots remains essentially unchanged. Aluminum ions in the imprinted sites form complexes with chlortetracycline, restricting the conformational rotation of the chlortetracycline molecule, thus continuously enhancing the inherent green fluorescence peak (at 500 nm). Establishing a linear relationship between the fluorescence intensity ratio and the chlortetracycline concentration allows for highly sensitive detection of chlortetracycline based on the fluorescence intensity changes of the test solution. Furthermore, under 365 nm UV illumination, the solution exhibits rich color changes, clearly showing a variation from red to orange-red, orange, yellow, yellow-green, and green as the chlortetracycline concentration increases.

[0050] The graph showing the change in fluorescence emission peak intensity of the non-blot fluorescent sensor with increasing chlortetracycline concentration and its color change under a 365nm UV lamp is shown below. Figure 5 .

[0051] Depend on Figure 5 It can be seen that, in comparison, when a non-imprinted fluorescent sensor lacking imprinting sites interacts with chlortetracycline within the same concentration range, the intensity change of the inherent green fluorescence peak (at 500 nm) of chlortetracycline is far less than that of a dual-emission molecular imprinted ratiometric fluorescent sensor. Therefore, under irradiation with a 365 nm ultraviolet lamp, the color change of the solution is not obvious.

[0052] 2. Selectivity of dual-emission molecularly imprinted ratiometric fluorescence sensors and non-imprinted fluorescence sensors

[0053] Take 100 μL of the molecularly imprinted polymer (Al) prepared earlier. 3+ 10 μL of the previously prepared silica-quantum dot composite nanoparticles (SiO2-QDs) and 10 μL of the pre-prepared silica-quantum dot composite nanoparticles were added to 880 μL of HEPES buffer (10 mmol / L, pH 6.5) to construct a dual-emission molecularly imprinted ratiometric fluorescence sensor. Then, 10 μL of 1 mmol / L solutions of chlortetracycline, tetracycline, oxytetracycline, doxycycline, ciprofloxacin, norfloxacin, threonine, glutathione, clindamycin, or sulfamethoxazole (final concentration 10 μmol / L) were added to the above dual-emission molecularly imprinted ratiometric fluorescence sensor (990 μL). The mixture was shaken well, and the fluorescence intensity of each sample was measured using a fluorescence spectrophotometer.

[0054] As a control, 100 μL of the previously prepared non-imprinted polymer (Al) was taken. 3+ 10 μL of the previously prepared silica-quantum dot composite nanoparticles (SiO2-QDs) and 10 μL of the silica-quantum dot composite nanoparticles were added to 880 μL of HEPES buffer (10 mmol / L, pH 6.5) to construct a non-blotted fluorescent sensor. Then, 10 μL of 1 mmol / L solutions of chlortetracycline, tetracycline, oxytetracycline, doxycycline, ciprofloxacin, norfloxacin, threonine, glutathione, clindamycin, or sulfamethoxazole (final concentration 10 μmol / L) were added to the above non-blotted fluorescent sensor (990 μL), mixed well, and the fluorescence intensity of each sample was measured using a fluorescence spectrophotometer.

[0055] The experimental results of the selectivity of the dual-emission molecularly imprinted ratiometric fluorescence sensor and the non-imprinted fluorescence sensor for different antibiotics and antibiotic analogs are shown in the figure. Figure 6 .

[0056] Depend on Figure 6 It can be seen that chlortetracycline exhibits the best fluorescence enhancement effect on the dual-emission molecularly imprinted ratiometric fluorescence sensor, followed by tetracycline and oxytetracycline. The fluorescence enhancement effect of these three on the dual-emission molecularly imprinted ratiometric fluorescence sensor is significantly higher than that of other antibiotics and antibiotic analogues. Because tetracycline, oxytetracycline, and chlortetracycline have similar chemical structures, tetracycline and oxytetracycline also show a high degree of fluorescence enhancement on the dual-emission molecularly imprinted ratiometric fluorescence sensor. This phenomenon indicates that the two-molecule imprinted polymer (Al... 3+ The cavity structure of MIPs plays a crucial role in fluorescence enhancement.

[0057] The above results demonstrate that the dual-emission molecularly imprinted ratiometric fluorescence sensor constructed in this invention has good selectivity for chlortetracycline.

[0058] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for fabricating a dual-emission molecularly imprinted ratiometric fluorescence sensor, characterized in that, Includes the following steps: (1) Preparation of silica-quantum dot composite nanoparticles: ultrapure water and silica solution were mixed, and then 3-aminopropyltriethoxysilane was added to obtain a mixed solution. After stirring for 30 min, N-acetyl-L-cysteine-modified cadmium telluride quantum dot solution was added and stirred for another 30 min. Then, ammonia monohydrate and tetraethoxysilane were added and stirred in the dark for 12±1 h to obtain silica-quantum dot composite nanoparticles. (2) Preparation of molecularly imprinted polymer: Salicylic amide was dissolved in ethanol, and ultrapure water, silica solution and 3-aminopropyltriethoxysilane were added in sequence to obtain a mixed solution. After stirring for 30 min, hexadecyltrimethylammonium bromide solution and sodium hydroxide solution were added and stirring was continued for 30 min. Then, ammonia monohydrate and tetraethoxysilane were added and stirring was continued for 3 h. Finally, aluminum chloride solution was added and stirring was carried out for 12 ± 1 h to obtain a molecularly imprinted polymer with salicylic amide template molecule. Salicylic amide was eluted to obtain the final molecularly imprinted polymer. (3) Construction of a dual-emission molecularly imprinted ratio fluorescence sensor: The molecularly imprinted polymer prepared in step (2) and the silica-quantum dot composite nanoparticles prepared in step (1) are mixed in HEPES buffer to construct a dual-emission molecularly imprinted ratio fluorescence sensor.

2. The method for preparing the dual-emission molecularly imprinted ratiometric fluorescence sensor according to claim 1, characterized in that, In steps (1) and (2), the method for preparing the silica solution is as follows: Ethanol and ultrapure water were mixed, and ammonia water with a mass concentration of 25%-28% was added. Tetraethoxysilane and ethanol were added dropwise using a constant pressure dropping funnel under the conditions of room temperature and magnetic stirring. The mixture was magnetically stirred for 12 hours at room temperature to obtain silica nanoparticles. After purification, the nanoparticles were dispersed in ultrapure water to obtain a silica solution.

3. The method for preparing a dual-emission molecularly imprinted ratiometric fluorescence sensor according to claim 1, characterized in that, In step (1), the method for preparing the N-acetyl-L-cysteine-modified cadmium telluride quantum dots is as follows: (i) Add ultrapure water, ethanol, sodium borohydride and tellurium powder to the reaction vessel and react for 4 hours under a water bath at 40°C to form a bright purple supernatant containing sodium tellurium hydride. Use this supernatant as a precursor for the synthesis of cadmium telluride quantum dots. (ii) Cadmium chloride and N-acetyl-L-cysteine ​​were dissolved in ultrapure water to obtain a mixed solution. After adjusting the pH to 9, nitrogen gas was purged to remove oxygen. Then, the newly prepared precursor was added to the mixed solution. The mixture was stirred at room temperature for 10 min and then heated under reflux for 1 h to obtain N-acetyl-L-cysteine-modified cadmium telluride quantum dots.

4. The method for preparing a dual-emission molecularly imprinted ratiometric fluorescence sensor according to claim 1, characterized in that, In step (1), the ratio of ultrapure water, silica solution, 3-aminopropyltriethoxysilane, N-acetyl-L-cysteine-modified cadmium telluride quantum dot solution, ammonia monohydrate and tetraethoxysilane is 13 mL: 400 μL: 10 μL: 2 mL: 100 μL: 100 μL.

5. The method for fabricating a dual-emission molecularly imprinted ratiometric fluorescence sensor according to claim 1, characterized in that, In step (2), the ratio of salicylamide, ethanol, ultrapure water, silica solution, 3-aminopropyltriethoxysilane, hexadecyltrimethylammonium bromide, sodium hydroxide, ammonia monohydrate, tetraethoxysilane and aluminum chloride is 10 mg: 0.5 mL: 13 mL: 400 μL: 15 μL: 0.1 mmol: 0.05 mmol: 120 μL: 120 μL: 1 mmol.

6. The method for preparing a dual-emission molecularly imprinted ratiometric fluorescence sensor according to claim 1, characterized in that, In step (3), the method for constructing the dual-emission molecularly imprinted ratiometric fluorescence sensor is as follows: (i) Disperse the molecularly imprinted polymer obtained in step (2) into ultrapure water; (ii) Disperse the silica-quantum dot composite nanoparticles prepared in step (1) into an amount of ultrapure water equal to that in step (i); (iii) The molecularly imprinted polymer dispersed in ultrapure water and the silica-quantum dot composite nanoparticles were mixed in HEPES buffer at a volume ratio of 10:

1.

7. A dual-emission molecularly imprinted ratiometric fluorescence sensor, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the dual-emission molecularly imprinted ratio fluorescence sensor of claim 7 in the detection of chlortetracycline.

Images