Ratio fluorescent probe for detecting tetracycline, test strip and preparation method and application thereof

Abstract

The application belongs to the technical field of analysis and detection, and particularly relates to a ratio fluorescent probe for detecting tetracycline, a test strip and a preparation method and application thereof. The ratio fluorescent probe preparation method provided by the application comprises the following steps: preparing an AIE polymer based on an MBH reaction; and combining the AIE polymer with AMP and a rare earth ion at room temperature to form a blue-fluorescent AIE-ICP composite material. The AIE polymer synthesized based on the MBH reaction is used as a host, the AMP is used as a ligand, and the rare earth ion is used as a central ion to synthesize the AIE-ICP composite material with strong blue fluorescence. The beta-diketone structure of tetracycline forms a stable complex with the rare earth ion, and the absorbed energy is effectively transferred to the rare earth ion through the "antenna effect", and then the characteristic fluorescence of the rare earth ion is enhanced, so that the concentration of tetracycline in the environment is determined through the ratio-type fluorescent analysis method, and the method has strong specificity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of analytical detection, and particularly relates to a ratio fluorescent probe for detecting tetracycline, a test strip and a preparation method and application thereof. BACKGROUND

[0002] In the livestock breeding industry, feeders often add antibiotics to the feed of livestock and poultry to prevent them from being infected with diseases. However, livestock and poultry can only absorb a small amount of antibiotics, especially tetracycline. The excess tetracycline is excreted with feces outside the animal body, thereby causing great harm to the soil environment and other ecological systems. At the same time, long-term consumption of animal-derived foods containing tetracycline drug residues can cause damage to the liver and kidney functions of the human body, and easily cause an increase in drug-resistant bacteria in the body and intestinal flora imbalance. The abuse of antibiotics has always threatened the survival and development of human beings, and has now become a new type of environmental pollutant. Its pollution problem has gradually attracted people's attention, so the detection of residual antibiotics has become a hot and difficult point in the chemical field. Therefore, it is of important theoretical and practical significance to select a specific and efficient method to detect the concentration of residual tetracycline in the environment.

[0003] At present, the methods for detecting tetracycline mainly include electrochemical biosensing, colorimetry and fluorescence. However, the implementation of simple optical strategies for tetracycline detection has not achieved great success. Compared with electrochemical biosensing and colorimetry, fluorescence has the advantages of fast response, simple operation, high sensitivity and low cost, and has attracted more and more research interest. For example, Chinese Patent Application CN110441280A discloses a method for detecting tetracycline and clenbuterol by using a carbon dot-rhodamine B dual-fluorescence system ratio fluorescent probe. The method uses phenylboric acid and 3-aminopropyl triethoxysilane as carbon and silicon sources to synthesize boron-silicon doped fluorescent carbon dots by a hydrothermal method. The synthesized boron-silicon doped fluorescent carbon dots and rhodamine B form a dual-fluorescence system, which produces three emission wavelengths of 414 nm, 578 nm and 670 nm. A dual-ratio fluorescent probe detection system is constructed by using rhodamine B as a fluorescent reference and the dual-wavelength of boron-silicon doped fluorescent carbon dots and the reference fluorescence. Tetracycline and clenbuterol produce linear fluorescence quenching and fluorescence sensitization effects on the dual-ratio fluorescent probe system, respectively, thereby establishing a ratio fluorescent probe detection method for tetracycline and clenbuterol. However, the method has a relatively complicated pretreatment operation, which is not conducive to popularization and application.

[0004] Therefore, there is an urgent need to develop a fluorescent detection method with good selectivity, anti-interference, stability and rapidness for sensitively and selectively detecting tetracycline. SUMMARY

[0005] In order to solve the problems in the prior art, the purpose of the present application is to provide a ratio fluorescent probe for detecting tetracycline, a test strip and a preparation method and application thereof. The ratio fluorescent probe for detecting tetracycline provided by the present application realizes visual quantitative detection of tetracycline, greatly reduces the interference of instruments, environmental matrices and the like on the detection results, has high sensitivity in the detection process, is simple in operation process, and is low in toxicity of materials, so that the whole detection process is environmentally friendly, safe and convenient.

[0006] The technical scheme of the present application is:

[0007] A preparation method of a ratio fluorescent probe for visual quantitative detection of tetracycline, comprising the following steps:

[0008] (1) preparing an AIE polymer with AIE luminescence phenomenon based on an MBH reaction;

[0009] (2) combining the AIE polymer with AMP and a rare earth ion at room temperature to form an AIE-ICP composite material with blue fluorescence.

[0010] The present application synthesizes an AIE-ICP composite material with strong blue fluorescence by taking the AIE polymer synthesized based on the MBH reaction as a main body, taking adenosine monophosphate (AMP) as a ligand, and taking a rare earth ion as a central ion. Tetracycline can form a stable complex with the rare earth ion in the AIE-ICP composite material through the beta-diketone structure of tetracycline, and can effectively transfer the absorbed energy to the rare earth ion through the “antenna effect”, thereby enhancing the characteristic fluorescence of the rare earth ion. The concentration of tetracycline in the environment is specifically determined by a ratio fluorescent analysis method (the ratio fluorescent probe indicates the content of the measured substance by the ratio of two fluorescence signals, overcomes the defect that a single fluorescent probe is easily disturbed, and amplifies the detection signal).

[0011] Further, the preparation method of the ratio fluorescent probe for visual quantitative detection of tetracycline comprises the following steps:

[0012] S1: synthesis of the AIE polymer: taking Boc-protected tert-butyl acetate and a luminescent group compound with an aggregation-induced emission phenomenon as monomers, and performing a room temperature reaction under the catalysis of DABCO in a low-polarity solvent to obtain the AIE polymer;

[0013] S2: mixing the AIE polymer, adenosine monophosphate and a rare earth ion obtained in step S1 at room temperature and shaking uniformly to obtain a milky white turbid liquid; centrifugally washing the milky white turbid liquid with deionized water, adding deionized water to the milky white turbid liquid after the washing is completed, and mixing uniformly to obtain the AIE-ICP composite material.

[0014] Compared with AIE small molecules, the AIE polymer prepared in the application as a new type of fluorescent material has good film-forming property, processability, mechanical strength and functional composite property, thereby being able to meet diversified application requirements. In addition, from the structural point of view, the existing AIE polymer is usually formed by covalent connection of AIE units, however, the performance of the AIE polymer of the application is not a simple superposition or stacking on the basis of small molecules, but shows more excellent performance, such as synergistic amplification effect, etc., so that it has a unique advantage in the application in the related field. On the other hand, lanthanide metals have important applications in the field of fluorescence detection due to their unique fluorescence characteristics, large stokes shift and long fluorescence lifetime, etc. Under ultraviolet light excitation, their adjacent chromophores can form f-f or f-d energy transfer systems, so that the Ln-ICP has strong visible fluorescence emission. In the system, the rare earth ion is the luminescent center, and adenosine-5'-monophosphate (AMP) is used as a ligand, and the system has specific responsiveness to the -diketone structure of tetracycline, thereby constructing a unique fluorescence sensing platform.

[0015] However, since the measurement depends on the change of the fluorescence intensity of the rare earth ion, i.e. the lanthanide ion, the detection result is easily disturbed by the fluctuation caused by environmental or instrumental factors, thereby causing the problem of low accuracy and precision of the measurement result. Fortunately, the inventors found in the research and development process that the selection of Eu 3+ as the central ion can effectively improve the selectivity of the system to tetracycline, because Eu 3+ has a large stokes shift and a long fluorescence lifetime, and the sharp line emission band produced by the forbidden parity f-f transition can effectively reduce the interference of the environment and the instrument on the measurement result. 3+ The ion complexing of tetracycline and Eu 3+ can transfer the excitation energy to Eu 3+ , so that Eu 3+ emits sensitization (this process is called "antenna effect"), thereby greatly reducing the interference of the instrument, the environment and the matrix on the measurement result.

[0016] Therefore, the rare earth ion in the application is preferably Eu 3+ .

[0017] Further, the molar ratio of the t-butyl acetate and the luminescent group compound is 1:1.

[0018] The molar ratio of the AIE polymer, the adenine ribonucleotide and the rare earth ion is 1:1:1.

[0019] Further, in the synthesis of the AIE polymer, the t-butyl acetate protected by Boc and the luminescent group compound having the aggregation-induced emission phenomenon are used as monomers, the room temperature reaction is carried out under the catalysis of DABCO in the low-polarity solvent dichloromethane, the AIE polymer is obtained by sedimentation with n-hexane and vacuum drying.

[0020] Further, the application also provides a ratio fluorescent probe prepared by the preparation method of the ratio fluorescent probe.

[0021] Further, the application also provides an AIE-ICP test strip for visualizing quantitative detection of tetracycline, which contains the AIE-ICP composite material synthesized in step (2). The AIE-ICP test strip is obtained by immersing a filter paper piece in the AIE-ICP composite material synthesized in step (2) above, taking it out and drying.

[0022] In addition, the application also provides an application of the ratio fluorescent probe in detection of tetracycline.

[0023] The application also provides a method for detecting tetracycline, comprising the following steps:

[0024] Step A: preparing an AIE polymer with AIE luminescence phenomenon based on MBH reaction;

[0025] Step B: combining the AIE polymer with AMP and rare earth ions at room temperature to form an AIE-ICP composite material with blue fluorescence;

[0026] Step C: adding tetracycline solutions with different concentrations into the AIE-ICP composite material obtained in step B to measure the fluorescence ratio of the AIE-ICP composite material (I 466.4 ) and the complex (I 616 ) of tetracycline and rare earth ions, and obtain a linear relationship curve between the I 466.4 / I 616 ratio and the tetracycline concentration;

[0027] Alternatively, the AIE-ICP test strip is immersed in tetracycline solutions with different concentrations for reaction, the AIE-ICP test strip is taken out, the color RGB value of the AIE-ICP test strip is measured, and a linear relationship curve between the color RGB value of the AIE-ICP test strip and the tetracycline concentration is obtained.

[0028] Step D: dropping a sample solution to be tested into the AIE-ICP composite material obtained in step B to measure the fluorescence ratio of the complex of the AIE-ICP composite material and the sample solution to be tested and rare earth ions;

[0029] Alternatively, the AIE-ICP test strip is immersed in a sample solution to be tested for reaction, the AIE-ICP test strip is taken out, and the color RGB value of the AIE-ICP test strip is measured.

[0030] Further, the volume ratio of the tetracycline solutions with different concentrations to the AIE-ICP composite material in step C is 1:1; and the AIE-ICP composite material (I466.4 ) with tetracycline and rare earth ions (I 616 ) fluorescence ratio.

[0031] Preferably, the present application provides a preparation method of the ratio fluorescent probe and a detection method of quantitatively detecting tetracycline, and the steps are as follows:

[0032] Step 1: preparation of polymer with AIE luminescence phenomenon based on MBH reaction;

[0033] Step 2: combination of AIE polymer with AMP and Eu 3+ at room temperature to form AIE-ICP composite material with blue fluorescence;

[0034] Step 3: adding tetracycline with different concentrations into the AIE-ICP composite material of step 2 to form a double-ratio fluorescent probe, and determining the fluorescence ratio of AIE-ICP composite material (I 466.4 ) with tetracycline and rare earth europium ion (Eu 3+ ) to form stable complex Eu-TC (I 616 ); tetracycline can form stable complex (Eu-TC) with rare earth europium ion (Eu 3+ ) through its own -diketone structure, and effectively transfer the absorbed energy to Eu 3+ , which produces characteristic red fluorescence at 616 nm, and the ratio of probe fluorescence intensity (I 466.4 / I 616 ) changes with the concentration of tetracycline, and the change of the ratio of probe fluorescence intensity causes obvious change of fluorescence color, thereby realizing visual detection and obtaining the linear relationship curve of I 466.4 / I 616 ratio and tetracycline concentration;

[0035] Step 4: combination of sample solution to be detected with AIE-ICP composite material, and tetracycline contained in the sample to be detected can form stable complex (Eu-TC) with rare earth europium ion (Eu 3+ ) through its own -diketone structure, and the fluorescence ratio of AIE-ICP composite material (I 466.4 ) with tetracycline and rare earth europium ion (Eu 3+ ) to form stable complex Eu-TC (I 616 ) is determined during detection, so as to obtain the concentration of tetracycline contained in the sample to be detected.

[0036] Further, the specific steps are as follows:

[0037] Step 1: The synthesis of AIE polymer is based on the Morita-Baylis-Hillman reaction (MBH) mechanism. Boc-protected MBH tert-butyl acetate and a luminescent group compound with aggregation-induced emission (AIE) are used as monomers in a molar ratio of 1:1. The reaction is carried out at room temperature for 48 hours under the catalysis of DABCO in a low polar solvent (such as dichloromethane). After precipitation with n-hexane and vacuum drying, AIE polymer with strong fluorescence is obtained.

[0038] Step 2: The AIE-ICP composite material was prepared using a one-pot method. The AIE polymer synthesized in Step 1 was used as the main component, adenosine monophosphate (AMP) as the ligand, and rare earth europium ions (Eu) were added. 3+ As the central ion, it was thoroughly mixed and shaken at a molar ratio of 1:1:1 at room temperature to obtain a milky white turbid liquid. After washing the milky white turbid liquid three times with deionized water by centrifugation, 1 mL of deionized water was added to the milky white turbid liquid and mixed for 5 minutes to obtain the AIE-ICP composite material.

[0039] Step 3: Tetracycline of different concentrations was added to the AIE-ICP composite material from Step 2. As the tetracycline concentration increased, the fluorescence of the AIE-ICP material gradually weakened, while the fluorescence of Eu... 3+ The fluorescence of the complex of the metal ion and tetracycline gradually increases, forming a ratiometric fluorescent probe. [AIE-ICP composite material (I 466.4 ) and tetracycline and rare earth europium ions (Eu) 3+ ) forms a stable complex Eu-TC(I) 616 The ratio of fluorescence intensity (1) 466.4 / 1 616 The fluorescence intensity of the two molecules is linearly related to the concentration of tetracycline, and tetracycline can be quantitatively determined based on the ratio of their fluorescence intensities.

[0040] Step 4: Cut out prototype filter paper pieces of the same size and shape, put them into the AIE-ICP composite material synthesized in Step 2, soak them for half an hour, then take them out and dry them in an oven to obtain AIE-ICP test strips; add different concentrations of tetracycline to the test strips, and as the concentration of tetracycline increases, the test strips show a color change from blue to red. The RGB value of the test strip color is linearly related to the tetracycline concentration, thus realizing the visual detection of tetracycline concentration.

[0041] The principle of this invention for quantitative detection of tetracycline is as follows: when the AIE-ICP composite material reacts with tetracycline, the central ion Eu...3+ The beta-diketone structure of tetracycline forms a stable complex, presents red fluorescence, and the blue fluorescence of the material itself is reduced due to the aggregation and Eu-TC doping, the ratio of the fluorescence intensity (1 466.4 / 1 616 ) changes with the change of the tetracycline concentration, and the change of the ratio of the fluorescence intensity of the probe causes the obvious change of the fluorescence color, so that the visual detection is realized.

[0042] Compared with the prior art, the ratio fluorescence probe for detecting tetracycline, the test strip and the preparation method and application thereof provided by the present application have the following advantages:

[0043] (1) The AIE polymer synthesized based on the MBH reaction is used as the main body, adenosine monophosphate (AMP) is used as the ligand, and the rare earth europium ion Eu 3+ is used as the central ion, so that the AIE-ICP composite material with strong blue fluorescence is synthesized; tetracycline can form a stable complex (Eu-TC) with the rare earth europium ion (Eu 3+ ) in the AIE-ICP composite material through the beta-diketone structure, and effectively transfer the absorbed energy to Eu 3+ through the "antenna effect", and then enhance the characteristic fluorescence of Eu 3+ , so that the ratio fluorescence analysis method can be established to specifically determine the concentration of tetracycline in the environment, and the interference of instruments, environmental matrices and the like on the detection result is greatly reduced. The Eu 3+ metal is selected as the central ion, so that the selectivity of the system to tetracycline can be effectively improved; and the unique spectral characteristics of Eu 3+ , such as long fluorescence lifetime, large stokes shift and sharp linear emission band generated by the forbidden parity f-f transition, result in that tetracycline can be combined with Eu 3+ ion, the excitation energy is transferred to Eu 3+ ion, and Eu 3+ ion emits sensitization.

[0044] (2) The detection method provided by the present application has high sensitivity and simple detection operation process, and the material is low in toxicity, so that the whole detection process is environmentally friendly, safe and convenient.

[0045] (3) When the concentration of tetracycline is 0.1-50 mg / L, the change of the ratio of the fluorescence intensity of the ratio fluorescence probe provided by the present application has a good linear relationship with the concentration of tetracycline, and can be used for quantitatively detecting the concentration of tetracycline. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1Scanning electron microscope (SEM) image (Figure A) and transmission electron microscope (TEM) image (Figure B) of AIE-ICP composite prepared in Example 3.

[0047] Figure 2 Infrared spectra of AIE-ICP composite prepared in Example 3 and after reaction with tetracycline;

[0048] Figure 3 Excitation-emission fluorescence spectra of AIE-ICP composite prepared in Example 3 (Figure A) and after reaction with tetracycline (Figure B).

[0049] Figure 4 Fluorescence intensity curve of AIE-ICP composite prepared in Example 3 after reaction with tetracycline of different concentrations.

[0050] Figure 5 Linear trend graph of ratio fluorescence probe prepared in Example 3 in determination of different concentrations of tetracycline. As shown in the figure, in the range of 0.1-50 mg / L, I 466.4 / I 616 The ratio is linearly related to the concentration of tetracycline, and the linear equation is ΔI = -7.313 log(c) + 20.352, R 2 = 0.9947.

[0051] Figure 6 Color change graph of test strip after addition of tetracycline of different concentrations.

[0052] Figure 7 Linear trend graph of color change of test strip and tetracycline concentration.

[0053] Figure 8 Fluorescence change trend graph of Comparative Example 1 after addition of tetracycline of different concentrations.

[0054] Figure 9 Fluorescence change trend graph of Comparative Example 2 after addition of tetracycline of different concentrations.

[0055] Figure 10 Fluorescence change trend graph of Comparative Example 3 after addition of tetracycline of different concentrations. DETAILED DESCRIPTION

[0056] The present application is further illustrated by the following description of specific embodiments, but this is not intended to limit the present application. Those skilled in the art can make various modifications or improvements to the present application according to the basic idea of the present application, as long as they do not deviate from the basic idea of the present application, and they are within the scope of protection of the present application.

[0057] In the following examples and comparative examples, the reagents not specifically mentioned are conventional reagents, which can be purchased from conventional reagent production and sales companies, and some raw material production companies and the like are as follows:

[0058] Tetracycline was purchased from Shanghai Macklin Biochemical Technology Co., Ltd.; 1,6-hexanediol diacrylate, benzaldehyde, 4,4'-(1,2-diphenyl ethylene-1,2-diyl) biphenol, 1,4-diazabicyclo and 4-dimethylamino pyridine were purchased from Shanghai Bide Co., Ltd.; di-tert-butyl dicarbonate was purchased from Guangzhou Rongman Biological Technology Co., Ltd.; adenosine-5'-triphosphate disodium salt hydrate (AMP), europium (III) chloride hexahydrate, sodium dodecyl sulfate (SDS) and 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0059] The AIE polymer was prepared in Example 1, and the steps were as follows:

[0060] Preparation of AIE polymer substrate:

[0061] SM1: In a 250 mL round-bottom flask, 21.2 g (200 mmol) of benzaldehyde was added, followed by 22.66 g (100 mmol) of 1,6-hexanediol diacrylate, 11.2 g (100 mmol) of 1,4-diazabicyclo [DABCO] was added under stirring, and finally 4 mL of methanol was added, and stirred at room temperature for 48 h or more. Stop the reaction, pour the reaction into 50 mL of water, extract with dilute hydrochloric acid and saturated sodium chloride (50 mL x 3), collect the organic phase, dry with anhydrous sodium sulfate, filter, collect the filtrate with a 250 mL single-neck round-bottom flask, and rotary evaporate. First, pass through a column with ethyl acetate: petroleum ether = 1:5 polar solvent, and finally collect the product with pure ethyl acetate to obtain a colorless liquid. The above 43.8 g (100 mmol) of product was dissolved in 60 mL of dichloromethane, and 54.5 g (250 mmol) of di-tert-butyl dicarbonate was added under stirring, 2.4 g (20 mmol) of 4-dimethylamino pyridine [DMAP] was added at 0°C, the reaction was tracked by TLC until the reaction was complete (about one and a half hours), then extracted with dichloromethane and dilute hydrochloric acid (100 mL x 2), dried with anhydrous sodium sulfate, filtered, rotary evaporated, then passed through a column with ethyl acetate: petroleum ether = 1:5 polar solvent to obtain a light yellow viscous liquid, which was the Boc-protected MBH tert-butyl acetate.

[0062] SM2: 4,4'-(1,2-diphenyl ethylene-1,2-diyl) biphenol was a commercially available product, which was purified by recrystallization and the like before reaction.

[0063] Preparation of AIE polymer: 127.8 mg (0.2 mmol) of SM1 and 45.7 mg (0.2 mmol) of SM2 were added to a 3 mL sample bottle, 2.2 mg (0.1 mmol) of DABCO and 1 mL of DCM solvent were added, and the reaction was carried out at room temperature for 24 h. After the reaction was completed, the pure AIE polymer was obtained by repeated sedimentation for 3 times with dichloromethane: n-hexane = 1:100.

[0064] Example 2 Preparation of AIE-ICP composite material, the steps are as follows:

[0065] The AIE polymer prepared in Example 1 was dissolved in 1 mL of tetrahydrofuran, mixed with an AMP solution (0.1 mol / L) solution in a ratio of 1:1, and then the solution was mixed with 1 mL of Eu 3+ solution (0.1 mol / L) and shaken well, and oscillated for 5 minutes. The centrifuge speed was adjusted to 10000 r / min, and centrifuged for 3 minutes. It should be noted that the centrifuge tube should be placed in balance. After centrifugation, the AIE-ICP composite material was obtained by washing with deionized water three times, and finally adding 1 mL of deionized water and mixing well to obtain a white turbid liquid.

[0066] Example 3 Preparation of a ratiometric fluorescent probe for visual detection of tetracycline, the steps are as follows:

[0067] 20 L of AIE-ICP composite material in Example 2 was added to a 96-well plate, 160 / 140 L of 1 mol / L HEPES buffer solution with pH of 7.4 and 0.086 mmol / L sodium dodecyl sulfate (SDS) solution were added, and mixed well for 2 minutes. Then 20 L of tetracycline with different concentrations (0, 10, 50, 100, 200, 250, 500, 750, 1000, 1500, 2000 mg / L) was taken, the volume in the hole was kept at 200 L, and the mixture was shaken well. Each concentration was operated in triplicate, and the fluorescence intensity ratio (I 466.4 / I 616 ) of the complex (I 466.4 ) of AIE-ICP composite material (I 616 ) with tetracycline and rare earth ions was measured at 616 nm.

[0068] The results are shown in Figure 1 , and it can be seen from the figure that the particle size of AIE-ICP composite material before reaction is large and irregular spherical, about 120 nm; after adding tetracycline, the overall particle size becomes small and irregular porous.

[0069] As shown in Figure 2 , 912 cm -1 and 1116 cm -1 , 1244 cm -1Symmetric and anti-symmetric stretching vibration peaks of the phosphate moiety of AMP, respectively, after the addition of tetracycline, the symmetric stretching vibration peak characteristic peak almost disappeared, anti-symmetric stretching vibration peak red shift to 1046cm -1 and 1186cm -1 , indicating that the AIE-ICP network structure may be destroyed, tetracycline with β-diketone structure and central ion Eu 3+ form a stable complex.

[0070] As shown in Figure 3 , the left graph is the fluorescence excitation-emission spectrum of AIE-ICP composite material, the maximum emission wavelength is at 380nm; the right graph is the fluorescence excitation-emission spectrum of AIE-ICP composite material after adding tetracycline with a concentration of 50mg / L, new fluorescence emission peaks appear at 580nm and 616nm.

[0071] As shown in Figure 4 , with the increase of tetracycline concentration, the fluorescence at 466.4nm is quenched, and the fluorescence at 616nm is enhanced, thus establishing a dual-ratio fluorescence probe detection method.

[0072] As shown in Figure 5 , the fluorescence intensity decreases in a parabolic manner with the increase of tetracycline concentration, and the logarithm of tetracycline concentration and the fluorescence intensity show a linear trend when the concentration of tetracycline added is 1-500mg / L, the linear equation is: ΔI=-7.313log(c)+20.352, R 2 =0.9947.

[0073] Example 4: Preparation of test paper strip for visual detection of tetracycline, the steps are as follows:

[0074] Take a piece of quantitative filter paper, cut it into small round pieces of uniform size with a puncher, and immerse it in the AIE-ICP composite material prepared in Example 2. Take out the filter paper piece and put it in a 60℃ oven to dry, add 1 to 2 drops of tetracycline with different concentrations (0, 10, 50, 100, 200, 500, 1000, 2000mg / L) on the dried filter paper piece, and wait for 20 minutes at room temperature. Then put the filter paper piece in a 60℃ oven to dry, and prepare the test paper strip for visual detection of tetracycline. Observe the color change of the test paper strip under ultraviolet lamp irradiation, and detect the color intensity of the test paper piece with the color picker of a smartphone or computer, record the RGB value of the test paper strip, and calculate the color intensity D value by the following formula through the Euclidean distance model (D).

[0075] After calculation, the numerical value is obtained, and a linear trend graph is drawn according to the change of the numerical value and the concentration of tetracycline. The results are shown in Figure 6 , 7 .

[0076]

[0077] R, G, and B represent the color intensity values ​​of the three primary colors of red, green, and blue, respectively. In the formula: R0, G0, and B0 are the color intensity values ​​of the blank; Ri, Gi, and Bi are the color intensity values ​​of the sample.

[0078] like Figure 6 As shown, the test strip changes from blue to orange-red as the tetracycline concentration increases. The luminescent insoluble particles are AIE polymers, and tetracycline gradually destroys their structure, causing the blue luminescence to weaken.

[0079] like Figure 7 As shown, the color intensity D value of the system decreases with increasing tetracycline concentration. Within the concentration range of 1-500 mg / L, the logarithm of the tetracycline concentration shows a linear trend with the color intensity D value, and the linear equation is: D = -66.188log(c) + 384.75, R 2 =0.9924.

[0080] Comparative Example 1

[0081] Comparative Example 1 is similar to Example 3, except that:

[0082] Synthesis of Eu / AMP ICP material: No AIE polymer was added during the synthesis process. Instead, 1 mL of 0.1 mol / L AMP solution and 1 mL of 0.1 mol / L Eu were added. 3+ Mix the solution well, centrifuge at 10000 r / min for 3 minutes, wash three times with deionized water, and finally add 1 mL of deionized water and mix well to obtain a milky white turbid liquid.

[0083] The fluorescence trend graphs after adding different concentrations of tetracycline to the material of Comparative Example 1 are shown below. Figure 8 As shown, without the addition of AIE polymer, fluorescence only increases linearly at 580 nm and 616 nm. Compared to the dual-ratio fluorescent probe, the detection is more susceptible to external interference, resulting in decreased sensitivity.

[0084] Comparative Example 2

[0085] Comparative Example 2 is similar to Example 3, except that:

[0086] Synthesis of TPE-DOH@Eu / AMP ICP material: During the synthesis process, 1 mL of 0.1 mol / L AMP solution was added and mixed with 1 mL of 1,2-bis(4-hydroxybenzene)-1,2-diphenylene dissolved in tetrahydrofuran. Then, 1 mL of 0.1 mol / L AMP solution was added. 3+The solution was mixed well, centrifuged at 10000 r / min for 3 minutes, washed with deionized water for three times, and finally mixed with 1 mL of deionized water to obtain a milky white turbid liquid.

[0087] After adding different concentrations of tetracycline into the material of Comparative Example 2, the fluorescence change trend chart is shown in Figure 9 After adding other small molecules with AIE phenomenon, fluorescence quenching appeared at 446 nm and fluorescence enhancement appeared at 621 nm, but the fluorescence decline trend at 446 nm was not linear, which was considered to be caused by poor dispersibility of AIE small molecules, and the destruction of AIE structure was not uniform after adding different concentrations of tetracycline, so it was difficult to detect quantitatively by double-ratio fluorescence.

[0088] Comparative Example 3

[0089] Comparative Example 3 is similar to Example 3, except that:

[0090] Synthesis of AIE@Tb / AMP ICP material: 1 mL of 0.1 mol / L AMP solution and 1 mL of AIE polymer dissolved in tetrahydrofuran were mixed, 1 mL of 0.1 mol / L Tb 3+ solution was added and mixed well, then centrifuged at 10000 r / min for 3 minutes, washed with deionized water for three times, and finally mixed with 1 mL of deionized water to obtain a milky white turbid liquid.

[0091] After adding different concentrations of tetracycline into the material of Comparative Example 3, the fluorescence change trend chart is shown in Figure 10 After replacing europium metal ions with terbium metal ions, terbium was difficult to form a stable complex with tetracycline with β-diketone structure, so no new fluorescence peak appeared at 580 nm and 616 nm, and the change of single-sided fluorescence intensity was more susceptible to external interference, and the sensitivity decreased.

[0092] Test Example Four Content Detection of Tetracycline

[0093] 1. Test object: Longtan Village river water

[0094] 2. Test method: 10 mL (1 mL 100 mg / L, 2000 mg / L, 4000 mg / L standard solution was added to 9 mL river water sample) river water was filtered through a 0.22 μm water phase filter, and then passed through a cation exchange resin. 20 μL of the AIE-ICP composite material obtained in Example 2 was added to a 96-well plate, 140 μL of 1 mol / L HEPES buffer solution with pH 7.4 was added, and 20 μL of 0.086 mmol / L sodium dodecyl sulfate (SDS) solution was added, mixed for 2 minutes, then 20 μL of treated river water sample was added, the volume in the well was kept at 200 L, and the mixture was shaken thoroughly. Three parallel groups were prepared. The fluorescence intensity ratio of the complex of AIE-ICP composite material (I 466.4 ) and tetracycline and rare earth ions (I 616 ) was determined at 616 nm. 466.4 616

[0095] 3. Test results:

[0096] The Longtan village river water sample was determined, and no tetracycline residue was found. The sample was determined for recovery rate at three concentrations, and the results are shown in Table 1. The average recovery rate was %, and from the measured data, the recovery rate of each spiked concentration was basically consistent, the recovery was complete, and the method was reliable.

[0097] Table 1: Recovery rate of spiked sample

[0098]

[0099] The above examples only illustratively explain the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical idea disclosed by the present application should be covered by the claims of the present application.​​

Claims

1. A method for preparing a ratiometric fluorescent probe for visualizing quantitative detection of tetracycline, characterized in that, Comprising the following steps: (1) Preparation of AIE polymer with AIE luminescence phenomenon based on MBH reaction, wherein the AIE polymer is prepared by reaction of SM1 prepared from 4,4'-(1,2-diphenyl ethylene-1,2-diyl) diphenol as SM2 and 1,6-hexanediol diacrylate and benzaldehyde as raw materials; (2) The AIE polymer is combined with adenine ribonucleotide and Eu 3+ The AIE-ICP composite material forms blue fluorescence at room temperature.

2. The method of preparing a ratiometric fluorescent probe for visualizing the quantitative detection of tetracycline according to claim 1, wherein, Comprising the following steps: ​ S1: Synthesis of AIE polymer: Boc-protected tert-butyl acetate and 4,4'-(1,2-diphenyl ethylene-1,2-diyl) diphenol as monomers, catalyzed by DABCO at room temperature under low-polarity solvent conditions, to obtain AIE polymer; S2: mixing the AIE polymer, adenine ribonucleotide and Eu 3+ Mixing and shaking at room temperature, a milky turbidity liquid was obtained; the milky turbidity liquid was washed by centrifugation with deionized water, and after the washing was completed, deionized water was added to the milky turbidity liquid, and mixed to obtain an AIE-ICP composite material.

3. The method for preparing a ratiometric fluorescent probe for the visual quantitative detection of tetracycline as described in claim 2, characterized in that, The molar ratio of the tert-butyl acetate and 4,4'-(1,2-diphenyl ethene-1,2-diyl) biphenol is 1:1; the molar ratio of the AIE polymer, adenine ribonucleotide and Eu 3+ is 1:1:

1.

4. The method of preparing a ratiometric fluorescent probe for visualizing and quantitatively detecting tetracycline according to claim 2, wherein, In the synthesis of the AIE polymer: Boc-protected tert-butyl acetate and 4,4'-(1,2-diphenyl ethylene-1,2-diyl) diphenol as monomers, catalyzed by DABCO at room temperature under low-polarity solvent dichloromethane conditions, precipitated with n-hexane, and vacuum dried to obtain AIE polymer. ​ 5. A ratio fluorescent probe prepared by the method of any one of claims 1-4.

6. An AIE-ICP test strip for visualizing quantitative detection of tetracycline, characterized in that, The AIE-ICP test strip contains the AIE-ICP composite material synthesized in step (2) of the method of any one of claims 1-4.

7. The ratio fluorescent probe of claim 5 for use in detecting tetracycline.

8. A method of detecting tetracycline, characterized by, Comprising the following steps: Step A: Different concentrations of tetracycline solution were added to the AIE-ICP composite material of any one of claims 1-4, and the fluorescence ratio of the AIE-ICP composite material to the tetracycline and Eu 3+ complex was determined to obtain the linear relationship curve of the I 466.4 / I 616 ratio and the tetracycline concentration. Alternatively, the AIE-ICP test strip for visualizing quantitative detection of tetracycline of claim 6 is immersed in different concentrations of tetracycline solution for reaction, the AIE-ICP test strip is taken out, the color RGB value of the AIE-ICP test strip is measured, and a linear relationship curve of the color RGB value of the AIE-ICP test strip and the concentration of tetracycline is obtained; Step B: drop the sample solution to be tested into the AIE-ICP composite material, and measure the fluorescence ratio of the complex of AIE-ICP composite material and Eu 3+ . Alternatively, after the AIE-ICP test strip is immersed in the sample solution to be tested for reaction, the AIE-ICP test strip is taken out, and the color RGB value of the AIE-ICP test strip is measured.

9. The method of detecting tetracycline according to claim 8, wherein, The volume ratio of the different concentrations of tetracycline solution to the AIE-ICP composite material in step A is 1:1.

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