Triemissive ratio molecularly imprinted fluorescent sensor, preparation method and application thereof

Abstract

The application discloses a three-emission ratio molecular imprinting fluorescent sensor and a preparation method and application thereof, and belongs to the technical field of chemical sensors. The three-emission ratio molecular imprinting fluorescent sensor is obtained by mixing blue fluorescent imprinting microspheres, green fluorescent imprinting microspheres and red fluorescent imprinting microspheres in a buffer solution according to a volume ratio of 5:5:1, each of the imprinting microspheres has a core-shell structure, each takes salicylamide as a virtual template, takes carbon dots as a fluorescent detection signal, takes bovine serum albumin as a sensitizing agent of doxycycline, takes CdTe quantum dots as a fluorescent detection signal, and is imprinted on the surface of SiO2 nanoparticles through a sol-gel method. The application has the advantages that the three-emission ratio molecular imprinting fluorescent sensor is creatively constructed, the fluorescent color change is rich, and an effective idea is provided for rapid and visual identification of doxycycline.

Description

TECHNICAL FIELD

[0001] The present application relates to a fluorescent sensor and its preparation method and application, in particular to a three-emission ratio molecular imprinting fluorescent sensor and its preparation method and application, and belongs to the technical field of chemical sensors. BACKGROUND

[0002] Doxycycline belongs to the tetracycline class of broad-spectrum antibiotics, which can act on gram-positive bacteria or gram-negative bacteria and many pathogenic bacteria resistant to cell wall inhibitor antibacterial agents. Doxycycline is commonly used as an antibiotic, mainly used in aquaculture and livestock breeding. The continuous accumulation of doxycycline in water will destroy the balance of the ecological system and be transmitted in the food chain through biological enrichment, thereby causing harm to the entire ecological environment and human health. Therefore, it is necessary to develop a sensitive, rapid and efficient method for detecting trace amounts of doxycycline.

[0003] Common doxycycline analysis methods mainly include chromatographic methods such as high-performance liquid chromatography and liquid chromatography-mass spectrometry, and other methods such as enzyme-linked immunoassay and biological methods. Chromatographic methods can give accurate analysis, but generally take a long time and require complex sample pretreatment and professional personnel operation. Biological methods and enzyme-linked immunoassay are relatively simple compared to chromatographic methods, but require a high variety of reagents, have high costs, are easily variable, and are greatly affected by complex matrix interference. Fluorescence analysis methods have rapidly developed in the fields of chemical sensing and biological analysis due to their strong specificity, high sensitivity, simple operation, and short time consumption. Ratio fluorescent sensors have attracted widespread attention due to their effective improvement of signal-to-noise ratio and reliable quantitative analysis. Currently, ratio fluorescent sensors for doxycycline analysis are mostly single / dual-emission mode, with a narrow color change window and a not wide enough color visual range. Therefore, methods for designing three-emission molecular imprinting fluorescent sensors for rapid and high-sensitivity detection of doxycycline have attracted much attention. Molecular imprinting polymers have rapidly developed in the fields of environment and life due to their high selectivity, simple preparation, good stability, and low cost. Compared with natural or other methods for preparing numerous recognition materials, molecular imprinting polymers provide a new idea for trace analysis and detection of doxycycline through specific recognition of target molecules. SUMMARY

[0004] The purpose of the present application is to provide a three-emission ratio molecular imprinting fluorescent sensor and its preparation method and application (rapid visual detection of doxycycline).

[0005] In order to achieve the above-mentioned target, the technical scheme adopted by the present application is as follows:

[0006] A three-emission ratio molecular imprinting fluorescent sensor is obtained by mixing blue fluorescent imprinting microspheres, green fluorescent imprinting microspheres and red fluorescent imprinting microspheres in a buffer solution at a volume ratio of 5:5:1, wherein:

[0007] Blue fluorescent imprinting microspheres: having a core-shell structure, with salicylamide as a virtual template, with carbon dots as a fluorescent detection signal, imprinting on the surface of SiO2 nanoparticles by sol-gel method;

[0008] Green fluorescent imprinting microspheres: having a core-shell structure, with salicylamide as a virtual template, with bovine serum albumin as a sensitizing agent for doxycycline, imprinting on the surface of SiO2 nanoparticles by sol-gel method;

[0009] Red fluorescent imprinting microspheres: having a core-shell structure, with salicylamide as a virtual template, with CdTe quantum dots as a fluorescent detection signal, imprinting on the surface of SiO2 nanoparticles by sol-gel method.

[0010] A preparation method of the aforementioned three-emission-ratio molecular imprinting fluorescent sensor, comprising the following steps:

[0011] (1) Preparation of blue fluorescent imprinting microspheres: dissolve salicylamide in water, continuously stir and add APTES and SiO2 nanoparticles, continue to stir the mixture, then add carbon dots, stir the mixture in the dark, then add NH3·H2O and TEOS, and stir the mixture in the dark overnight to obtain an imprinting polymer, elute the obtained imprinting polymer with ethanol / acetonitrile solution for 3 times to obtain blue fluorescent imprinting microspheres;

[0012] (2) Preparation of green fluorescent imprinting microspheres: dissolve salicylamide in water, continuously stir and add APTES and SiO2 nanoparticles, stir the mixture, then add bovine serum albumin and CTAB, continue to react, then add NH3·H2O and TEOS, and stir the mixture in the dark to obtain an imprinting polymer, elute the obtained imprinting polymer with ethanol / acetonitrile solution for 3 times to obtain green fluorescent imprinting microspheres;

[0013] (3) Preparation of red fluorescent imprinting microspheres: dissolve salicylamide in water, continuously stir and add APTES and SiO2 nanoparticles, continue to stir the mixture, then add CdTe quantum dots, stir the mixture in the dark, then add NH3·H2O and TEOS, and stir the mixture in the dark overnight to obtain an imprinting polymer, elute the obtained imprinting polymer with ethanol / acetonitrile solution for 3 times to obtain red fluorescent imprinting microspheres;

[0014] (4) Mixing: disperse the obtained blue fluorescent imprinting microspheres, green fluorescent imprinting microspheres and red fluorescent imprinting microspheres into equal amounts of water respectively, then mix them in a buffer according to a volume ratio of 5:5:1.

[0015] Preferably, in step (1), the amount ratio of salicylamide, APTES, SiO2 nanoparticles, carbon dots, NH3·H2O and TEOS is 10 mg:10 μL:8 mg:4 mL:20 μL:20 μL.

[0016] Preferably, in step (2), the amount ratio of salicylamide, APTES, SiO2 nanoparticles, bovine serum albumin, CTAB, NH3·H2O and TEOS is 10 mg:5 μL:8 mg:65 mg:0.2 mmol:50 μL:50 μL.

[0017] Preferably, in step (3), the amount ratio of salicylamide, APTES, SiO2 nanoparticles, CdTe quantum dots, NH3·H2O and TEOS is 10 mg:5 μL:8 mg:5 mL:50 μL:50 μL.

[0018] Preferably, in step (1), step (2) and step (3), the ethanol / acetonitrile solution is mixed by ethanol and acetonitrile according to 8:2 volume ratio.

[0019] Preferably, in step (4), the buffer is 10 mmol / L, pH 8.5 Tris-HCl buffer.

[0020] The aforementioned three-emission ratio molecularly imprinted fluorescent sensor is applied to detection of doxycycline.

[0021] The present application has the advantages of:

[0022] (1) The bovine serum albumin involved in the present application not only enhances the fluorescence intensity of doxycycline as a sensitizer, but also participates in the preparation of green fluorescent imprinting microspheres as an auxiliary functional monomer.

[0023] (2) The present application selects salicylamide similar in structural properties to doxycycline as a virtual template, uses the blue fluorescence peak quenching and dynamic quenching mechanism of the red fluorescence peak of the three-emission ratio molecularly imprinted fluorescent sensor as a fluorescence signal source, and creatively constructs the three-emission ratio molecularly imprinted fluorescent sensor based on the principle of the sensitization of bovine serum albumin and doxycycline to the enhancement of the inherent green fluorescence peak of doxycycline, which provides an effective idea for rapid and visual identification of doxycycline with rich fluorescence color changes.

[0024] (3) The three-emission ratio molecularly imprinted fluorescent sensor prepared by the present application has the advantages of simple operation, rapidness, high selectivity, high sensitivity, good reliability and strong practicability in detecting doxycycline. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1is a schematic diagram of the preparation process of the three-emission ratio molecular imprinting fluorescent sensor;

[0026] Figure 2 is an electron microscope scanning result diagram of SiO2 nanoparticles, different color fluorescent imprinting microspheres, and different color fluorescent non-imprinting microspheres;

[0027] Figure 3 is a diagram of the change of the fluorescence emission peak intensity of the three-emission ratio molecular imprinting fluorescent sensor with the increase of the concentration of doxycycline;

[0028] Figure 4 is a diagram of the change of the fluorescence emission peak intensity of the three-emission ratio non-imprinting fluorescent sensor with the increase of the concentration of doxycycline;

[0029] Figure 5 is a diagram of the change of the fluorescence emission peak intensity of the three-emission ratio molecular imprinting fluorescent sensor with the increase of the concentration of doxycycline;

[0030] Figure 6 is a diagram of the selectivity experiment results of the three-emission ratio molecular imprinting fluorescent sensor and the three-emission ratio non-imprinting fluorescent sensor to different antibiotic analogs. DETAILED DESCRIPTION

[0031] The application will be specifically described below in combination with the drawings and specific embodiments.

[0032] I. Synthesis of fluorescent nanomaterials

[0033] 1. Synthesis of carbon dots

[0034] The temperature of the air drying oven was increased to 225℃, 0.5g of tris-hydroxymethyl aminomethane and 2g of citric acid were weighed into a small beaker, and the small beaker was placed in the air drying oven. After about 3min, the solid in the small beaker began to melt, and after about 7min, the solution in the small beaker turned orange yellow. The small beaker was taken out and 10mL of ultrapure water was added thereto. Under continuous stirring, the pH was adjusted to neutral with 1M sodium hydroxide solution, and dialysis was performed for 24h (dialysis bag: MWCO 3500Da) to remove unreacted substances and small molecular impurities, to obtain carbon dots, which were stored at 4℃ in the dark.

[0035] 2. Synthesis of CdTe quantum dots

[0036] A precursor was formed by thoroughly mixing 68.4 mg of cadmium chloride and 63 μL of mercaptoacetic acid in 75 mL of ultrapure water, and adjusting the pH to 9.0. After stirring for 30 min under nitrogen, 1 mL of freshly prepared aqueous sodium borohydride solution (40 mg of sodium borohydride and 38.3 mg of tellurium powder reacted in a 40 °C ethanol / water solution (1.5 mL / 0.5 mL) for 4 h) was quickly injected into the precursor with stirring. The solution was heated and refluxed for 36 h to obtain red CdTe quantum dots capped with mercaptoacetic acid.

[0037] II. Preparation of fluorescent imprinting microspheres of different colors

[0038] Reference Figure 1 Using salicylamide (similar in structural properties to doxycycline) as a virtual template, carbon dots and CdTe quantum dots as fluorescent detection signals, and bovine serum albumin as a sensitizing agent for doxycycline, fluorescent imprinting microspheres with a core-shell structure, including blue fluorescent imprinting microspheres, red fluorescent imprinting microspheres, and bovine serum albumin-sensitized doxycycline green fluorescent imprinting microspheres, were prepared by sol-gel method on the surface of SiO2 nanoparticles.

[0039] 1. Preparation of blue fluorescent imprinting microspheres

[0040] 10 mg of salicylamide was weighed into 15 mL of water, and 10 μL of 3-aminopropyl triethoxysilane (APTES) and 8 mg of SiO2 nanoparticles were added under continuous stirring. The mixture was stirred for 0.5 h to perform a pre-polymerization process, then 4 mL of carbon dots was added, and the mixture was stirred in the dark for 0.5 h. After that, 20 μL of ammonia water (NH3·H2O) and 20 μL of tetraethyl orthosilicate (TEOS) were added, and the mixture was stirred in the dark overnight to obtain an imprinting polymer. The obtained imprinting polymer was eluted 3 times with an ethanol / acetonitrile (8:2, v / v) solution to remove salicylamide, resulting in blue fluorescent imprinting microspheres. The blue fluorescent imprinting microspheres emitted blue fluorescence under the irradiation of 410 nm ultraviolet light, and the blue fluorescence was quenched after the addition of doxycycline. The obtained blue fluorescent imprinting microspheres were dispersed in 2 mL of water for further experiments.

[0041] As a control, blue fluorescent non-imprinting microspheres were synthesized using the same steps but without the addition of salicylamide.

[0042] 2. Preparation of green fluorescent imprinting microspheres

[0043] Take 10 mg of salicylamide and dissolve it in 15 mL of water, then add 5 μL of APTES and 8 mg of SiO2 nanoparticles under continuous stirring, stir the mixture for 20 min, then add 65 mg of auxiliary functional monomer bovine serum albumin and 1 mL of 0.2 mol / L CTAB, continue to react for 0.5 h, then add 50 μL of NH3·H2O and 50 μL of TEOS, and stir the mixture in the dark for 4 h to obtain the imprinted polymer. The obtained imprinted polymer is eluted 3 times with ethanol / acetonitrile (8:2, v / v) solution to remove salicylamide, and green fluorescent imprinted microspheres are obtained. The green fluorescent imprinted microspheres do not emit light under the irradiation of 485 nm visible light, but emit green fluorescence after the addition of doxycycline. The obtained green fluorescent imprinted microspheres are dispersed in 2 mL of water for further experiments.

[0044] As a control, green fluorescent non-imprinted microspheres are synthesized using the same steps but without the addition of salicylamide.

[0045] 3. Preparation of red fluorescent imprinted microspheres

[0046] Take 10 mg of salicylamide and dissolve it in 15 mL of water, then add 5 μL of APTES and 8 mg of SiO2 nanoparticles under continuous stirring, stir the mixture for 0.5 h to perform a pre-polymerization process, then add 5 mL of CdTe quantum dots, and stir the mixture in the dark for 0.5 h, then add 50 μL of NH3·H2O and 50 μL of TEOS, and stir the mixture in the dark overnight to obtain the imprinted polymer. The obtained imprinted polymer is eluted 3 times with ethanol / acetonitrile (8:2, v / v) solution to remove salicylamide, and red fluorescent imprinted microspheres are obtained. The red fluorescent imprinted microspheres emit red fluorescence under the irradiation of 626 nm visible light, and the red fluorescence is quenched after the addition of doxycycline. The obtained red fluorescent imprinted microspheres are dispersed in 2 mL of water for further experiments.

[0047] As a control, red fluorescent non-imprinted microspheres are synthesized using the same steps but without the addition of salicylamide.

[0048] III. Observation of the micro-morphology of SiO2 nanoparticles, imprinted microspheres and non-imprinted microspheres

[0049] A small amount of SiO2 nanoparticles and the previously prepared blue fluorescent imprinted microspheres, blue fluorescent non-imprinted microspheres, green fluorescent imprinted microspheres, green fluorescent non-imprinted microspheres, red fluorescent imprinted microspheres and red fluorescent non-imprinted microspheres are vacuum dried, and then scanned with a scanning electron microscope.

[0050] The results of the electron microscopy scanning of SiO2 nanoparticles, different color fluorescent imprinted microspheres and different color fluorescent non-imprinted microspheres are shown in Figure 2Wherein, A is the scanning electron microscopy result of SiO2 nanoparticles, B and C are the scanning electron microscopy results of blue fluorescent imprinted microspheres and blue fluorescent non-imprinted microspheres respectively, D and E are the scanning electron microscopy results of green fluorescent imprinted microspheres and green fluorescent non-imprinted microspheres respectively, F and G are the scanning electron microscopy results of red fluorescent imprinted microspheres and red fluorescent non-imprinted microspheres respectively.

[0051] It can be seen that: (1) SiO2 nanoparticles are near-spherical in morphology, have good dispersibility, and have an average diameter of about 100 nm; (2) both imprinted microspheres and non-imprinted microspheres have rough surfaces, and have an average diameter of about 120 nm, and there is no obvious difference in morphology and size between imprinted microspheres and non-imprinted microspheres. Figure 2

[0052] Four, preparation of a three-emission-ratio molecularly imprinted fluorescent sensor

[0053] The different color fluorescent imprinted microspheres prepared in the foregoing are mixed in a buffer solution by a post-imprinting mixing strategy, and a three-emission-ratio molecularly imprinted fluorescent sensor is prepared.

[0054] As a control, the same method is used, but the different color fluorescent imprinted microspheres are replaced by the different color fluorescent non-imprinted microspheres prepared in the foregoing, and a three-emission-ratio non-imprinted fluorescent sensor is prepared.

[0055] Four, detection of doxycycline using a three-emission-ratio molecularly imprinted fluorescent sensor

[0056] 1. Effect of concentration of doxycycline on detection

[0057] 100 μL of blue fluorescent imprinted microspheres, 100 μL of green fluorescent imprinted microspheres, and 20 μL of red fluorescent imprinted microspheres are taken respectively and added to 770 μL of Tris-HCl buffer solution (10 mmol / L, pH 8.5), and a three-emission-ratio molecularly imprinted fluorescent sensor is prepared.

[0058] As a control, 100 μL of blue fluorescent non-imprinted microspheres, 100 μL of green fluorescent non-imprinted microspheres, and 20 μL of red fluorescent non-imprinted microspheres are taken respectively and added to 770 μL of Tris-HCl buffer solution (10 mmol / L, pH 8.5), and a three-emission-ratio non-imprinted fluorescent sensor is prepared.

[0059] ​A series of different concentrations of doxycycline solution was prepared with pure water, and the final concentrations of doxycycline were 0.1 μmol / L, 0.5 μmol / L, 1.0 μmol / L, 2.5 μmol / L, 3.0 μmol / L, 5.0 μmol / L, 7.5 μmol / L, 10.0 μmol / L, 12.5 μmol / L, 15.0 μmol / L, 25.0 μmol / L, 30.0 μmol / L, 40.0 μmol / L, and 50.0 μmol / L, respectively.

[0060] 10 μL of doxycycline solution of different concentrations was added to the above-mentioned three-emission-ratio molecularly imprinted fluorescent sensor or three-emission-ratio non-imprinted fluorescent sensor, respectively, and after shaking, the fluorescence intensity of each sample was measured by a fluorescence instrument.

[0061] The change in the intensity of the fluorescence emission peak of the three-emission-ratio molecularly imprinted fluorescent sensor with the increase of the concentration of doxycycline is shown in Figure 3 From Figure 3 it can be seen that with the increase of the concentration of doxycycline, the intensity of the blue emission peak (410 nm) and the red emission peak (626 nm) of the three-emission-ratio molecularly imprinted fluorescent sensor gradually decreased, and the intensity of the green emission peak (485 nm) gradually increased.

[0062] The change in the intensity of the fluorescence emission peak of the three-emission-ratio non-imprinted fluorescent sensor with the increase of the concentration of doxycycline is shown in Figure 4 From Figure 4 it can be seen that with the increase of the concentration of doxycycline, the intensity of the blue emission peak (410 nm), the green emission peak (485 nm), and the red emission peak (626 nm) of the three-emission-ratio non-imprinted fluorescent sensor gradually decreased.

[0063] In addition, under the irradiation of a 365 nm ultraviolet lamp, it can be found by naked eye observation that:

[0064] (1) When the three-emission-ratio molecularly imprinted fluorescent sensor was used to detect doxycycline, with the increase of the concentration of doxycycline, the color of the solution changed from blue-violet to purple, pink, orange, yellow, and finally to green, and the color change was rich. It can be seen that by establishing a linear relationship between the change in the fluorescence intensity ratio and the concentration of doxycycline, and according to the change in the fluorescence intensity of the to-be-detected solution, high-sensitivity detection of doxycycline can be realized;

[0065] (2) When the three-emission-ratio non-imprinted fluorescent sensor was used to detect doxycycline, with the increase of the concentration of doxycycline, the color of the solution always presented blue-violet. It can be seen that the sensitivity of the three-emission-ratio non-imprinted fluorescent sensor to doxycycline was very low.

[0066] 2, The influence of the mixing ratio of different color fluorescent imprinting microspheres on detection

[0067] Take 100 μL blue fluorescent imprinting microspheres, 100 μL green fluorescent imprinting microspheres, 20 μL red fluorescent imprinting microspheres (volume ratio of 5:5:1) respectively, add to 770 μL Tris-HCl buffer (10 mmol / L, pH 8.5) to prepare three emission ratio molecular imprinting fluorescent sensor A.

[0068] Take 40 μL blue fluorescent imprinting microspheres, 100 μL green fluorescent imprinting microspheres, 20 μL red fluorescent imprinting microspheres (volume ratio of 2:5:1) respectively, add to 770 μL Tris-HCl buffer (10 mmol / L, pH 8.5) to prepare three emission ratio molecular imprinting fluorescent sensor B.

[0069] Take 120 μL blue fluorescent imprinting microspheres, 100 μL green fluorescent imprinting microspheres, 20 μL red fluorescent imprinting microspheres (volume ratio of 6:5:1) respectively, add to 770 μL Tris-HCl buffer (10 mmol / L, pH 8.5) to prepare three emission ratio molecular imprinting fluorescent sensor C.

[0070] Take 100 μL blue fluorescent imprinting microspheres, 120 μL green fluorescent imprinting microspheres, 20 μL red fluorescent imprinting microspheres (volume ratio of 5:6:1) respectively, add to 770 μL Tris-HCl buffer (10 mmol / L, pH 8.5) to prepare three emission ratio molecular imprinting fluorescent sensor D.

[0071] Prepare a series of doxycycline solutions with different concentrations. The final concentrations of doxycycline are 0.1 μmol / L, 0.5 μmol / L, 1.0 μmol / L, 2.5 μmol / L, 3.0 μmol / L, 5.0 μmol / L, 7.5 μmol / L, 10.0 μmol / L, 12.5 μmol / L, 15.0 μmol / L, 25.0 μmol / L, 30.0 μmol / L, 40.0 μmol / L, and 50.0 μmol / L, respectively.

[0072] Take 10 μL of doxycycline solution with different concentrations respectively, add to the three emission ratio molecular imprinting fluorescent sensor A, three emission ratio molecular imprinting fluorescent sensor B, three emission ratio molecular imprinting fluorescent sensor C, or three emission ratio molecular imprinting fluorescent sensor D, mix well, and then measure the fluorescence intensity of each sample using a fluorescence instrument.

[0073] The changes of fluorescence emission peak intensity of each three emission ratio molecular imprinting fluorescent sensor with the increase of doxycycline concentration are shown in the following table: Figure 5Among them, A is the change of the fluorescence emission peak intensity of the three-emission ratio molecular imprinting fluorescent sensor A with the increase of doxycycline concentration, B is the change of the fluorescence emission peak intensity of the three-emission ratio molecular imprinting fluorescent sensor B with the increase of doxycycline concentration, C is the change of the fluorescence emission peak intensity of the three-emission ratio molecular imprinting fluorescent sensor C with the increase of doxycycline concentration, and D is the change of the fluorescence emission peak intensity of the three-emission ratio molecular imprinting fluorescent sensor D with the increase of doxycycline concentration.

[0074] Depend on Figure 5 It can be seen that with the increase of doxycycline concentration, the intensities of the blue emission peak (410 nm) and red emission peak (626 nm) of triple emission ratio molecular imprinting fluorescent sensor A, triple emission ratio molecular imprinting fluorescent sensor B, triple emission ratio molecular imprinting fluorescent sensor C and triple emission ratio molecular imprinting fluorescent sensor D gradually decreased, while the intensity of the green emission peak (485 nm) gradually increased. The intensity changes of the fluorescence emission peaks of the four triple emission ratio molecular imprinting fluorescent sensors are the same.

[0075] However, under 365nm ultraviolet light, it can be found by naked eye observation:

[0076] (1) When the volume ratio of blue fluorescent imprinted microspheres, green fluorescent imprinted microspheres, and red fluorescent imprinted microspheres is 5:5:1, as the concentration of doxycycline increases, the color of the solution can be clearly observed to change from blue-purple to purple, pink, orange, yellow, and finally to green, with rich color changes. The green fluorescence peak of the sensor gradually increases, and green is the end color. The red and blue fluorescence peaks are quenched, which is an important reason for the color change.

[0077] (2) When the volume ratio of blue fluorescent imprinted microspheres, green fluorescent imprinted microspheres, and red fluorescent imprinted microspheres was 2:5:1, the initial color of the solution was rose red. As the concentration of doxycycline increased, the color of the solution changed from rose red to pink, yellow, and finally to green. The presence of blue fluorescence was almost unobservable, and the early appearance of the green color of the solution had no significant difference from the dual emission assay.

[0078] (3) When the volume ratio of blue fluorescent imprinted microspheres, green fluorescent imprinted microspheres, and red fluorescent imprinted microspheres is 6:5:1, as the concentration of doxycycline increases, the color of the solution changes from purple to gray, with no obvious transition and a single color.

[0079] (4) When the volume ratio of blue fluorescent imprinted microspheres, green fluorescent imprinted microspheres, and red fluorescent imprinted microspheres was 5:6:1, as the concentration of doxycycline increased, the color of the solution quickly changed from purple to green, showing a purple-white-green color change, with fewer transition colors and repeated end point colors.

[0080] It can be seen that the mixing ratio of blue fluorescent imprinting microspheres, green fluorescent imprinting microspheres and red fluorescent imprinting microspheres has a significant influence on the detection of doxycycline, and the optimal mixing volume ratio of blue fluorescent imprinting microspheres, green fluorescent imprinting microspheres and red fluorescent imprinting microspheres is 5:5:1.

[0081] 3. Selectivity of the three-emission ratio molecular imprinting fluorescent sensor

[0082] 100 μL of blue fluorescent imprinting microspheres, 100 μL of green fluorescent imprinting microspheres and 20 μL of red fluorescent imprinting microspheres (volume ratio of 5:5:1) were taken respectively and added to 770 μL of Tris-HCl buffer (10 mmol / L, pH 8.5) to prepare a three-emission ratio molecular imprinting fluorescent sensor, which was denoted as DMIPs.

[0083] As a control, 100 μL of blue fluorescent non-imprinting microspheres, 100 μL of green fluorescent non-imprinting microspheres and 20 μL of red fluorescent non-imprinting microspheres (volume ratio of 5:5:1) were taken respectively and added to 770 μL of Tris-HCl buffer (10 mmol / L, pH 8.5) to prepare a three-emission ratio non-imprinting fluorescent sensor, which was denoted as DNIPs.

[0084] A doxycycline solution with a concentration of 1 mmol / L, a tetracycline solution with a concentration of 1 mmol / L, an oxytetracycline solution with a concentration of 1 mmol / L, a chlortetracycline solution with a concentration of 1 mmol / L, a serine solution with a concentration of 1 mmol / L, a threonine solution with a concentration of 1 mmol / L, a glutathione solution with a concentration of 1 mmol / L, a clindamycin solution with a concentration of 1 mmol / L, a sulfadiazine solution with a concentration of 1 mmol / L, a sulfamerazine solution with a concentration of 1 mmol / L, a sulfanilamide solution with a concentration of 1 mmol / L, a sulfamethoxazole solution with a concentration of 1 mmol / L, a ciprofloxacin solution with a concentration of 1 mmol / L, an enoxacin solution with a concentration of 1 mmol / L, a Cd 2+ solution with a concentration of 1 mmol / L, a Pb 2+ solution with a concentration of 1 mmol / L and a Cu 2+ solution with a concentration of 1 mmol / L were prepared with pure water.

[0085] 10 μL of each prepared solution with a concentration of 1 mmol / L was taken and added to the above-mentioned three-emission ratio molecular imprinting fluorescent sensor (DMIPs) or three-emission ratio non-imprinting fluorescent sensor (DNIPs), and the final concentration of each solution was 10 μmol / L. After shaking, the fluorescence intensity of each sample was determined by a fluorescence instrument.

[0086] The selective experiment results of the three emission ratio molecular imprinting fluorescent sensors (DMIPs) and three emission ratio non-imprinting fluorescent sensors (DNIPs) on different antibiotic analogues are shown in Table 1 Figure 6 As shown in Table 1, the doxycycline has the best fluorescence enhancement effect on the three emission ratio molecular imprinting fluorescent sensors (DMIPs), followed by tetracycline, terramycin, aureomycin and other tetracycline antibiotics, which is much higher than other antibiotics and amino acids. Figure 6

[0087] Since the chemical structures of doxycycline, tetracycline, terramycin and aureomycin are similar, the fluorescence enhancement degree is also high, which can also explain that the cavity structure of the bovine serum albumin plays a key role in the fluorescence enhancement process.

[0088] The above results prove that the three emission ratio molecular imprinting fluorescent sensors (the volume ratio of blue fluorescent imprinting microspheres, green fluorescent imprinting microspheres and red fluorescent imprinting microspheres is 5:5:1) prepared by the present application have good selectivity for doxycycline detection.

[0089] In summary, the present application first prepares fluorescent imprinting microspheres of different colors (using salicylamide as a virtual template) by a sol-gel method, and then mixes the prepared fluorescent imprinting microspheres of different colors in a buffer solution by an imprinting mixing strategy to prepare a three emission ratio molecular imprinting fluorescent sensor. The blue fluorescent carbon dots, green fluorescent doxycycline and red fluorescent CdTe quantum dots in the three emission ratio molecular imprinting fluorescent sensor all serve as fluorescence response signals, and the fluorescence intensity decreases, increases and decreases, respectively, with the increase of the concentration of the target substance. The fluorescence intensity of the solution is determined by a fluorescence instrument (fluorescence spectrophotometer), the three emission peak intensities change to different degrees, and rich fluorescence color changes in the blue-violet, purple, pink, orange, yellow and green color ranges are produced. The corresponding relationship between the red-green-blue three-color fluorescence intensity ratio change and the doxycycline concentration can be used to realize the visual quantitative detection of doxycycline in a to-be-detected solution.

[0090] It should be noted that the above examples are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can be made by those of ordinary skill in the art. Here, all the embodiments cannot be exhausted. Any obvious changes or variations derived from the technical solutions of the present application are still within the protection scope of the present application.​

Claims

1. A triple emission ratio molecularly imprinted fluorescence sensor, characterized in that: The blue fluorescent imprinted microspheres, green fluorescent imprinted microspheres and red fluorescent imprinted microspheres are dispersed in equal amounts of water, and then mixed in a buffer solution at a volume ratio of 5:5:1, wherein: Blue fluorescent imprinted microspheres: They have a core-shell structure, use salicylamide as a virtual template, and carbon dots as fluorescence detection signals, and are imprinted on the surface of SiO2 nanoparticles via the sol-gel method. Green fluorescent imprinted microspheres: with a core-shell structure, using salicylamide as a virtual template and bovine serum albumin as a doxycycline sensitizer, are imprinted on the surface of SiO2 nanoparticles via a sol-gel method; Red fluorescent imprinted microspheres: have a core-shell structure, use salicylamide as a virtual template, and CdTe quantum dots as fluorescence detection signals, and are imprinted on the surface of SiO2 nanoparticles through the sol-gel method.

2. The method for preparing the triple emission ratio molecularly imprinted fluorescence sensor according to claim 1, characterized in that: The following steps are involved: (1) Preparation of blue fluorescent imprinted microspheres: salicylamide was dissolved in water, APTES and SiO2 nanoparticles were added under continuous stirring, the mixture was stirred, and then carbon dots were added. The mixture was stirred in the dark, and then NH3·H2O and TEOS were added. The mixture was stirred in the dark overnight to obtain an imprinted polymer. The imprinted polymer was eluted three times with ethanol / acetonitrile solution to obtain blue fluorescent imprinted microspheres. (2) Preparation of green fluorescent imprinted microspheres: salicylamide was dissolved in water, APTES and SiO2 nanoparticles were added under continuous stirring, bovine serum albumin and CTAB were added after stirring the mixture, and the reaction was continued. NH3·H2O and TEOS were then added and the mixture was stirred in the dark to obtain an imprinted polymer. The imprinted polymer was eluted three times with ethanol / acetonitrile solution to obtain green fluorescent imprinted microspheres. (3) Preparation of red fluorescent imprinted microspheres: salicylamide was dissolved in water, APTES and SiO2 nanoparticles were added under continuous stirring, the mixture was stirred, and then CdTe quantum dots were added. The mixture was stirred in the dark, and then NH3·H2O and TEOS were added. The mixture was stirred in the dark overnight to obtain an imprinted polymer. The imprinted polymer was eluted three times with ethanol / acetonitrile solution to obtain red fluorescent imprinted microspheres. (4) Mixing: The obtained blue fluorescent imprinted microspheres, green fluorescent imprinted microspheres, and red fluorescent imprinted microspheres were dispersed in equal amounts of water, and then mixed in a buffer solution at a volume ratio of 5:5:

1.

3. The preparation method according to claim 2, characterized in that In step (1), the usage ratio of salicylamide, APTES, SiO2 nanoparticles, carbon dots, NH3·H2O, and TEOS is 10 mg:10 μL:8 mg:4 mL:20 μL:20 μL.

4. The preparation method according to claim 2, characterized in that In step (2), the usage ratio of salicylamide, APTES, SiO2 nanoparticles, bovine serum albumin, CTAB, NH3·H2O and TEOS is 10 mg:5 μL:8 mg:65 mg:0.2 mmol:50 μL:50 μL.

5. The preparation method according to claim 2, characterized in that In step (3), the usage ratio of salicylamide, APTES, SiO2 nanoparticles, CdTe quantum dots, NH3·H2O and TEOS is 10 mg:5 μL:8 mg:5 mL:50 μL:50 μL.

6. The preparation method according to claim 2, characterized in that In step (1), step (2) and step (3), the ethanol / acetonitrile solution is prepared by mixing ethanol and acetonitrile in a volume ratio of 8:

2.

7. The preparation method according to claim 2, characterized in that In step (4), the buffer solution is 10 mmol / L Tris-HCl buffer solution with a pH of 8.

5.

8. Use of the triple emission ratio molecularly imprinted fluorescent sensor according to claim 1 in detecting doxycycline.

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