D-A type organic fluorescent small molecule, preparation method and application of D-A type organic fluorescent small molecule in detection of nitrofuran antibiotics

Abstract

The invention provides a D-A type organic fluorescent small molecule, a preparation method and application of the D-A type organic fluorescent small molecule in detection of nitrofuran antibiotics, and belongs to the technical field of fluorescent sensing. The D-A type organic fluorescent micromolecule is composed of 2-(2-aminophenyl) benzothiazole and an electron-donating group, the excitation spectrum of the D-A type organic fluorescent micromolecule is obviously overlapped with the absorption spectrum of nitrofuran antibiotics, and fluorescence quenching response can be shown through the inner filter effect; the trace nitrofuran antibiotics can be detected in a liquid phase in an ultrafast, high-sensitivity, excellent-selectivity and strong-anti-interference manner. According to the invention, a quantitative evaluation system can be constructed in combination with a smart phone, the RGB value of the image of the liquid-phase fluorescence sensor is output in real time, and rapid, accurate and visual quantitative detection of nitrofuran antibiotics in a real food sample or a water source is realized. In addition, the filter paper fluorescence sensor has the advantages of being portable and easy to prepare, and nitrofuran antibiotics can be rapidly identified on site in an actual scene.

Description

Technical Field

[0001] The invention relates to the field of fluorescence sensing technology, and in particular to a DA type organic fluorescent small molecule, a preparation method and application thereof in detecting nitrofuran antibiotics. Background Art

[0002] Veterinary drug residues caused by the misuse of antibiotics are a serious and widespread pollutant in food safety, posing a huge risk to human health and the environment. It is worth noting that nitrofuran antibiotics, as synthetic broad-spectrum antibiotics, were once widely used in the prevention and treatment of diseases in poultry and aquaculture. Veterinary drug residues can cause potential harm to various animals, plants and human health through the environment and food chain, leading to enhanced bacterial resistance in the body, causing allergic reactions or severe shock. Studies have further shown that excessive accumulation of nitrofuran antibiotics can cause side effects such as acute and chronic liver poisoning, prolonged blood coagulation time, carcinogenicity, and teratogenicity. In response to the serious health hazards caused by nitrofuran drug residues, many countries such as the European Union, the United States and China have formulated strict regulations to prohibit the use of nitrofuran drugs in animal-derived foods. However, driven by economic interests, the illegal abuse of nitrofuran antibiotics is still rampant. Therefore, the development of accurate and sensitive methods for the detection of nitrofuran antibiotic residues is crucial to protecting the environment and human health.

[0003] At present, the main detection methods of nitrofuran antibiotics include liquid chromatography-tandem mass spectrometry (LC-MS / MS), ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), high performance liquid chromatography-diode array detection (HPLC-DAD), enzyme-linked immunosorbent assay (ELISA), immunochromatographic assay (ICA), electrochemical assay, time-resolved fluorescence immunochromatography, surface enhanced Raman spectroscopy (SERS), etc. However, these methods generally have the disadvantages of fixed equipment, high cost, complex operation, poor portability, etc., which hinder further on-site real-time and visual detection.

[0004] Based on the characteristics of rapid response, high sensitivity and specificity, and easy integration into handheld devices, fluorescent sensors are considered more suitable for field applications. At present, the fluorescent material systems reported for the detection of nitrofuran antibiotics mainly include carbon dots (CDs), metal organic frameworks (MOFs), porous organic polymers (POPs), covalent organic frameworks (COFs), etc. However, these materials also face some inevitable disadvantages, such as the difficulty in obtaining CDs materials with uniform particle size and the complex purification process; the difficulty in synthesizing MOFs, POPs and COFs, the high cost of raw materials, and the need for ultrasonic pretreatment when used.

[0005] In contrast, organic fluorescent small molecules have the advantages of simple synthesis, clear structure, and good solubility, making them an ideal choice for fluorescent probes of nitrofuran antibiotics. In addition, fluorescent sensors loaded on filter paper are becoming more and more popular because of their low cost, portability, and environmental friendliness. However, there are few studies on organic fluorescent small molecules in antibiotic detection, and the sensing mechanism is still unclear. Therefore, it is necessary to develop more cost-effective and efficient organic fluorescent small molecules to detect the residues of nitrofuran antibiotics in actual environmental samples. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a DA-type organic fluorescent small molecule, a preparation method and its application in detecting nitrofuran antibiotics. The DA-type organic fluorescent small molecule provided by the present invention can realize ultrafast, highly sensitive and visualized fluorescence detection of nitrofuran antibiotics.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a DA-type organic fluorescent small molecule having a structure shown in Formula I:

[0009]

[0010] In Formula I, R is

[0011] Preferably, it has a structure shown in any one of Formula II to Formula VI:

[0012]

[0013] The present invention provides a method for preparing the above-mentioned DA type organic fluorescent small molecule, comprising the following steps:

[0014] A compound having a structure shown in formula A, a compound B, a catalyst, a solvent and an alkaline reagent are mixed and subjected to a Suzuki-Miyaura coupling reaction to obtain a DA-type organic fluorescent small molecule having a structure shown in formula I;

[0015] In formula A, X is Cl, Br or I;

[0016] The compound B has a structure shown in any one of formulas B1 to B11:

[0017]

[0018] In the structures represented by formulas B1 to B11, Y is a boronic acid group or a boronic ester group.

[0019] The present invention provides application of the DA type organic fluorescent small molecule in detecting nitrofuran antibiotics.

[0020] Preferably, the nitrofuran antibiotics include one or more of nitrofurazone, nitrofurantoin and furazolidone;

[0021] The nitrofuran antibiotics are nitrofuran antibiotics in food or water sources.

[0022] The present invention provides a liquid-phase fluorescence sensor, comprising a DA-type organic fluorescent small molecule and a preparation solvent, wherein the DA-type organic fluorescent small molecule is the above-mentioned DA-type organic fluorescent small molecule.

[0023] The present invention provides a method for quantitatively detecting nitrofuran antibiotics, comprising the following steps:

[0024] The sample to be tested is mixed with the liquid phase fluorescence sensor to obtain the fluorescence quenching rate;

[0025] Obtaining the concentration of nitrofuran antibiotics in the sample to be tested according to the fluorescence quenching rate and a predetermined standard curve;

[0026] The standard curve is a linear relationship curve between the concentration of nitrofuran antibiotics and the fluorescence quenching rate.

[0027] The present invention provides a visual quantitative detection method for nitrofuran antibiotics, comprising the following steps:

[0028] The sample to be tested is mixed with the liquid phase fluorescence sensor, and a fluorescent image of the resulting mixed liquid is obtained using a smartphone, and the RGB value of the fluorescent image is output in real time to obtain the rate of change of the G value;

[0029] The concentration of nitrofuran antibiotics in the sample to be tested is obtained according to the change rate of the G value and a predetermined standard curve.

[0030] The invention provides a filter paper fluorescence sensor, comprising filter paper and a fluorescent material loaded on the surface of the filter paper, wherein the fluorescent material comprises the above-mentioned DA type organic fluorescent small molecule.

[0031] The present invention provides a method for detecting nitrofuran antibiotics based on the filter paper fluorescence sensor, comprising the following steps:

[0032] The sample to be tested is added to the surface of the filter paper fluorescence sensor, the filter paper fluorescence sensor is irradiated with an ultraviolet light source, and the fluorescence color change of the filter paper fluorescence sensor is observed with the naked eye. If the fluorescence of the filter paper fluorescence sensor is completely quenched, it is determined that the sample to be tested contains nitrofuran antibiotics.

[0033] The present invention provides a DA-type organic fluorescent small molecule having a structure shown in Formula I. The entire molecular skeleton of the DA-type organic fluorescent small molecule provided by the present invention is composed of an electron acceptor (2-(2-aminophenyl)benzothiazole) and an electron donor (i.e., the R group in Formula I). ​​This type of DA-type organic fluorescent small molecule material has the following beneficial effects:

[0034] (1) Using 2-(2-aminophenyl)benzothiazole as the main luminescent building block and as an electron acceptor group, by introducing electron donor groups such as tetraphenylethylene and triphenylamine, an intramolecular charge transfer state (ICT) can be formed to construct a DA-type organic fluorescent small molecule. The degree of ICT can be effectively adjusted to regulate its absorption wavelength and excitation wavelength.

[0035] (2) 2-(2-Aminophenyl)benzothiazole has a rigid planar conjugated structure. After being covalently linked to an electron donor with a twisted configuration, the interaction between molecules is reduced, ensuring high luminescence efficiency, which is conducive to the efficient detection of nitrofuran antibiotics.

[0036] (3) After the 2-(2-aminophenyl)benzothiazole group is connected to the electron donor group, its absorption peak and excitation spectrum at the long wavelength will be red-shifted, increasing the degree of overlap with the absorption peak of nitrofuran antibiotics, thereby further improving the detection sensitivity and response time of the DA-type organic fluorescent small molecule to nitrofuran antibiotics.

[0037] (4) Among the DA-type organic fluorescent small molecules, P-BT3PCz can still maintain excellent optical properties at 40% water content, and can realize the fluorescence detection of nitrofuran antibiotics in a mixed system of water and organic solvents. At the same time, different pH conditions have little effect on its luminescence, and it has excellent photobleaching properties.

[0038] (5) Since the excitation spectrum of the organic small molecule fluorescent probe overlaps significantly with the absorption spectrum of nitrofuran antibiotics, and the fluorescence lifetime does not change with the addition of antibiotics, when the probe is excited by ultraviolet light, its excitation light will be absorbed by the nitrofuran antibiotics, and the fluorescence will be rapidly quenched, thereby realizing sensitive detection of instantaneous nitrofuran antibiotics.

[0039] (6) The DA-type organic fluorescent small molecule is used as a guest material to prepare a filter paper fluorescent sensor. Because there is a certain twist angle between the donor and the acceptor in the molecule, it helps to increase the molecular cavity and make it fully contact with the analyte, thereby improving its detection performance.

[0040] The present invention prepares a liquid phase fluorescence sensor and a filter paper fluorescence sensor for detecting nitrofuran antibiotics based on the above-mentioned DA-type organic fluorescent small molecules, which can realize ultrafast (3s), high sensitivity (LOD less than 57.85nM), excellent selectivity and strong anti-interference ability (28 interfering substances) fluorescence detection of nitrofuran (NFZ), nitrofurantoin (NFT) and furazolidone (FZD). The liquid phase fluorescence sensing system prepared by the present invention can realize accurate quantitative fluorescence detection of nitrofuran antibiotics in real food samples and water sources (shrimp, chicken breast, lake water, tap water). Further, the present invention can be combined with a smart phone to construct a quantitative evaluation system, which can output the RGB value of the fluorescence sensor image based on the DA-type organic fluorescent small molecule in real time, and realize rapid, accurate and visual quantitative detection of nitrofuran antibiotics in real food samples or water sources. In addition, the filter paper fluorescence sensor has the advantages of being portable and easy to prepare, and can quickly identify nitrofuran antibiotics on site in actual scenes. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The UV absorption spectrum and fluorescence emission spectrum of P-BT3PCz in water and N,N-dimethylformamide (volume ratio of 4:6);

[0042] Figure 2 The fluorescence emission spectrum of the solution after adding furacilin to the P-BT3PCz liquid phase fluorescence sensor and the fluorescence quenching rate-concentration linear standard curve;

[0043] Figure 3 The fluorescence emission spectrum of the solution after adding nitrofurantoin and the fluorescence quenching rate-concentration linear standard curve of the P-BT3PCz liquid phase fluorescence sensor;

[0044] Figure 4 The fluorescence emission spectrum of the solution after adding furazolidone and the fluorescence quenching rate-concentration linear standard curve of the P-BT3PCz liquid phase fluorescence sensor;

[0045] Figure 5 The relationship between the fluorescence intensity and time of the solution after adding different concentrations of nitrofuran antibiotics to the P-BT3PCz liquid phase fluorescence sensor;

[0046] Figure 6 It is a bar graph of the fluorescence quenching rate of P-BT3PCz liquid phase fluorescence sensor after adding different interfering substances;

[0047] Figure 7 It is a bar graph of the fluorescence quenching rate of P-BT3PCz liquid phase fluorescence sensor after adding mixed solutions of different interfering substances and nitrofuran antibiotics;

[0048] Figure 8The excitation and emission spectra of the P-BT3PCz liquid phase fluorescence sensor, the absorption spectrum of nitrofuran antibiotics, and the fluorescence lifetime spectra of the P-BT3PCz liquid phase fluorescence sensor after adding different concentrations of NFZ;

[0049] Fig. 9 Fluorescence images, RGB value analysis and standard curve of P-BT3PCz liquid phase fluorescence sensor after adding different concentrations of furacilin;

[0050] Fig.10 The results and comparison of the quantitative detection of nitrofuran antibiotics in real food samples (shrimp, chicken breast, lake water and tap water) by P-BT3PCz liquid phase fluorescence sensor by fluorescence method and RGB method respectively;

[0051] Fig.11 Visual qualitative detection results of nitrofuran antibiotics by filter paper fluorescence sensor prepared based on P-BT3PCz;

[0052] Fig.12 The fluorescence emission spectra of the solutions after adding nitrofuran antibiotics to the P-BTTPE liquid phase fluorescence sensor;

[0053] Fig.13 Fluorescence emission spectra of the solution after adding nitrofuran antibiotics to the P-BTTPA liquid phase fluorescence sensor;

[0054] Fig.14 The fluorescence emission spectra of the solution after adding nitrofuran antibiotics to the P-BTPCz liquid phase fluorescence sensor;

[0055] Fig.15 Fluorescence emission spectra of the solution after adding nitrofuran antibiotics to the P-BTDMA liquid phase fluorescence sensor. DETAILED DESCRIPTION

[0056] The present invention provides a DA-type organic fluorescent small molecule having a structure shown in Formula I:

[0057]

[0058] In Formula I, R is

[0059] In the present invention, the DA-type organic fluorescent small molecule has a structure shown in any one of Formula II to Formula VI:

[0060]

[0061] The DA type organic fluorescent small molecule provided by the present invention has good solubility in organic solvents, and is conducive to the preparation of liquid phase fluorescent sensors and filter paper fluorescent sensors.

[0062] The present invention provides a method for preparing the above-mentioned DA type organic fluorescent small molecule, comprising the following steps:

[0063] A compound having a structure shown in formula A, a compound B, a catalyst, a solvent and an alkaline reagent are mixed and subjected to a Suzuki-Miyaura coupling reaction to obtain a DA-type organic fluorescent small molecule having a structure shown in formula I;

[0064] In formula A, X is Cl, Br or I;

[0065] The compound B has a structure shown in any one of formulas B1 to B11:

[0066]

[0067] In the structures represented by formulas B1 to B11, Y is a boronic acid group or a boronic ester group.

[0068] Unless otherwise specified, the raw materials used in the present invention are all commercially available.

[0069] In the present invention, the catalyst preferably includes an organic palladium catalyst, and the organic palladium catalyst is preferably one or both of tetrakis(triphenylphosphine)palladium and bis(triphenylphosphine)palladium dichloride, and more preferably tetrakis(triphenylphosphine)palladium.

[0070] In the present invention, the alkaline agent preferably includes one or both of potassium carbonate and potassium phosphate, more preferably potassium carbonate; the alkaline agent is preferably added in the form of an aqueous solution, and the concentration of the aqueous solution is preferably 1.5 to 2.5 mol / L, more preferably 2 mol / L.

[0071] In the present invention, the molar ratio of the compound having the structure represented by formula A, compound B, catalyst and alkaline agent is preferably 1: (1-5): (0.04-0.1): (8-30), and more preferably 1: 1.2: 0.05: (10-20).

[0072] The present invention has no special requirements for the solvent, and an organic solvent well known to those skilled in the art can be used. As a specific embodiment of the present invention, the organic solvent is preferably a mixed solvent of toluene and ethanol, and the volume ratio of toluene and ethanol in the mixed solvent is preferably (2-5):1, more preferably 3:1; the present invention has no special requirements for the amount of the organic solvent, as long as it can completely dissolve the reaction raw materials.

[0073] In the present invention, the mixing method is preferably:

[0074] (a) mixing a compound having a structure represented by formula A, compound B and an alkaline agent to obtain a first mixed system;

[0075] (b) freezing and evacuating the first mixed system in sequence, and adding a catalyst and an organic solvent thereto under a protective atmosphere to obtain a second mixed system;

[0076] (c) freezing and evacuating the second mixed system in sequence.

[0077] In the present invention, the freezing in (b) and (c) is preferably liquid nitrogen freezing; the present invention has no special restrictions on the vacuuming method in (b) and (c), and the vacuuming method well known in the art can be used; in (b) and (c), freezing and vacuuming are performed in sequence as one operation, and the operation is repeated, and the number of repetitions is preferably 3 times, the time for a single freezing is preferably 10 minutes, and the time for a single vacuuming is preferably 5 minutes. The present invention adopts the above-mentioned feeding sequence and the pretreatment method of freezing and vacuuming to remove oxygen in the reaction system as much as possible to avoid oxidation deactivation of palladium catalysts, providing favorable conditions for Suzuki-Miyaura coupling reaction.

[0078] In the present invention, the Suzuki-Miyaura coupling reaction is preferably carried out under a protective atmosphere, and the protective atmosphere is preferably argon. In the present invention, the temperature of the Suzuki-Miyaura coupling reaction is 85 to 90°C, and the time is preferably 24 to 48 hours; as a specific embodiment of the present invention, the temperature of the Suzuki-Miyaura coupling reaction can be 85°C, 88°C or 90°C, and the time can be 24 hours, 36 hours or 48 hours.

[0079] After the Suzuki-Miyaura coupling reaction, the present invention preferably performs post-treatment on the obtained coupling reaction solution, and the post-treatment preferably comprises the following steps:

[0080] After the coupling reaction solution was cooled to room temperature, it was extracted with water and dichloromethane, and the organic phase was collected;

[0081] The organic phase is dried and rotary evaporated in sequence to obtain a crude product;

[0082] The crude product is purified by column chromatography, and the obtained purified product is recrystallized to obtain the pure product of the DA type organic fluorescent small molecule.

[0083] In the present invention, the drying agent used in the drying is preferably anhydrous magnesium sulfate; the role of the rotary evaporation is to remove the organic solvent. In the present invention, the eluent used in the column chromatography purification is preferably dichloromethane and petroleum ether, and the volume ratio of dichloromethane and petroleum ether is preferably (1-5): (1-20), more preferably (1-3): (1-9); the solvent used in the recrystallization is preferably a good solvent and a poor solvent, the good solvent is preferably dichloromethane, and the poor solvent is preferably hexane. The specific operation of the recrystallization is preferably: after using a small amount of dichloromethane to just dissolve the purified product, hexane is added until the pure product is precipitated, that is, the DA type organic fluorescent small molecule pure product.

[0084] The present invention provides application of the DA type organic fluorescent small molecule in detecting nitrofuran antibiotics.

[0085] In the present invention, the nitrofuran antibiotics include one or more of nitrofurazone, nitrofurantoin and furazolidone.

[0086] In the present invention, the nitrofuran antibiotics are preferably nitrofuran antibiotics in food or water source, and the food is preferably animal-derived food. As a specific embodiment of the present invention, the food is shrimp or chicken breast, and the water is lake water or tap water.

[0087] The invention provides a liquid phase fluorescence sensor, comprising the above-mentioned DA type organic fluorescent small molecule and a preparation solvent.

[0088] In the present invention, the preparation solvent is preferably a mixture of water and an organic solvent; in the present invention, the water is preferably ultrapure water, and the organic solvent preferably includes one of acetone, tetrahydrofuran, anhydrous ethanol, N,N-dimethylformamide and dimethyl sulfoxide, and is more preferably N,N-dimethylformamide; in the present invention, the volume ratio of water to organic solvent is preferably (0-9): (1-10), and is more preferably 4:6. The present invention can ensure the fluorescence intensity of DA-type organic fluorescent small molecules by controlling the composition of the preparation solvent.

[0089] In the present invention, the concentration of DA-type organic fluorescent small molecules in the liquid phase fluorescent sensor is preferably 0.5-2 μM, more preferably 1 μM. The present invention has no special requirements for the preparation method of the liquid phase fluorescent sensor, and the DA-type organic fluorescent small molecules can be directly dissolved in the preparation solvent.

[0090] The present invention provides a quantitative detection method for the above-mentioned nitrofuran antibiotics, comprising the following steps:

[0091] Mixing the sample to be tested with the liquid phase fluorescence sensor to obtain the fluorescence quenching rate;

[0092] Obtaining the concentration of nitrofuran antibiotics in the sample to be tested according to the fluorescence quenching rate and a predetermined standard curve;

[0093] The standard curve is a linear relationship curve between the concentration of nitrofuran antibiotics and the fluorescence quenching rate.

[0094] In the present invention, the nitrofuran antibiotics include one or more of nitrofurazone, nitrofurantoin and furazolidone.

[0095] In the present invention, the sample to be tested is preferably food or water source; the food is preferably animal-derived food. As a specific embodiment of the present invention, the food is shrimp or chicken breast, and the water is lake water or tap water. In the present invention, before the detection, the present invention preferably pre-treats the sample to be tested. When the sample to be tested is solid, the pre-treatment preferably includes the following steps:

[0096] The crushed sample to be tested is mixed with an organic solvent, subjected to ultrasonic extraction and centrifugation to obtain a supernatant;

[0097] The supernatant was filtered.

[0098] In the present invention, the organic solvent is preferably one of acetone, tetrahydrofuran, anhydrous ethanol, N,N-dimethylformamide and dimethyl sulfoxide, more preferably N,N-dimethylformamide; the mass ratio of the sample to be tested to the organic solvent is preferably 1:5. In the present invention, the power of the ultrasonic extraction is preferably 800W, the time is preferably 25 to 35 minutes, more preferably 30 minutes. In the present invention, the centrifugal speed is preferably 7000 to 8000 rpm, more preferably 8000 rpm; the time is preferably 4 to 5 minutes, more preferably 5 minutes.

[0099] In the present invention, the filtration is preferably organic membrane filtration, and the pore size of the organic membrane is preferably 0.45 μm.

[0100] In the present invention, when the sample to be tested is a liquid, the pretreatment preferably includes the following steps:

[0101] The sample to be tested is centrifuged and filtered.

[0102] In the present invention, the centrifugal speed is preferably 7000-8000 rpm, more preferably 8000 rpm; the time is preferably 4-5 min, more preferably 5 min. In the present invention, the filtration is preferably water-based membrane filtration, and the pore size of the water-based membrane is preferably 0.45 μm.

[0103] The sample to be tested is mixed with the above-mentioned liquid phase fluorescence sensor to obtain the fluorescence quenching rate. In the present invention, the fluorescence quenching rate = 1-I / I 0 , where I0 is the initial fluorescence intensity of the organic small molecule probe solution, and I is the fluorescence intensity of the solution after adding nitrofurazone, nitrofurantoin or furazolidone.

[0104] The present invention has no special requirements for the detection of fluorescence intensity, and a fluorescence intensity detection method familiar to those skilled in the art can be used. In the embodiment of the present invention, an RF-6000 fluorescence spectrophotometer is preferably used to measure the fluorescence intensity, and the excitation wavelength is preferably determined according to the structure of the organic small molecule fluorescent probe.

[0105] In the present invention, the standard curve is preferably obtained by a fluorescence calibration experiment. In the present invention, the method for obtaining the standard curve preferably comprises the following steps:

[0106] Provide gradient solutions of nitrofuran antibiotics with known concentrations;

[0107] The nitrofuran antibiotic solutions with known gradient concentrations are used as samples to be tested, and the nitrofuran antibiotic solutions with known gradient concentrations are mixed with liquid phase fluorescence sensors respectively to obtain fluorescence quenching rates corresponding to nitrofuran antibiotic solutions with different concentrations;

[0108] The nitrofuran antibiotic solution and its corresponding fluorescence quenching rate are linearly fitted to obtain a linear relationship curve between the concentration of the nitrofuran antibiotic and the fluorescence quenching rate, specifically a fluorescence quenching rate-nitrofurazone concentration standard curve, a fluorescence quenching rate-nitrofurantoin concentration standard curve, and a fluorescence quenching rate-furazolidone concentration standard curve.

[0109] In the present invention, the concentration range of the nitrofurazone standard solution is preferably 1 to 100 μM, the linear range of the fluorescence quenching rate-nitrofurazone concentration standard curve is preferably 1 to 10 μM, and the detection limit is preferably 57.85 nM; the concentration range of the nitrofurantoin standard solution is preferably 1 to 100 μM, the linear range of the fluorescence quenching rate-nitrofurantoin concentration standard curve is preferably 1 to 10 μM, and the detection limit is preferably 63.45 nM; the concentration range of the furazolidone standard solution is preferably 1 to 100 μM, the concentration range of the fluorescence quenching rate-furazolidone concentration standard curve is preferably 1 to 10 μM, and the detection limit is preferably 65.38 nM.

[0110] The present invention provides a visual quantitative detection method for nitrofuran antibiotics, comprising the following steps:

[0111] The sample to be tested is mixed with the above-mentioned liquid phase fluorescence sensor, and a fluorescence image of the resulting mixed solution is obtained using a smart phone and the RGB value of the fluorescence image is output in real time to obtain the rate of change of the G value;

[0112] The concentration of nitrofuran antibiotics in the sample to be tested is obtained according to the change rate of the G value and a predetermined standard curve.

[0113] In the present invention, the nitrofuran antibiotics include one or more of nitrofurazone, nitrofurantoin and furazolidone.

[0114] In the present invention, the types of samples to be tested and the pretreatment methods are the same as above and will not be described in detail here.

[0115] In the present invention, the G value change rate = 1-G / G 0 , where G 0 is the initial G value of the fluorescence pattern, and G is the G value of the fluorescence pattern after adding the sample to be tested.

[0116] In the present invention, the standard curve is preferably a G value change rate-nitrofurazone concentration standard curve, a G value change rate-nitrofurantoin concentration standard curve or a G value change rate-furazolidone concentration standard curve.

[0117] In the present invention, the linear detection range of the standard curve is preferably 1 to 10 μM; the detection limit of nitrofurazone is preferably 62.53 nM; the detection limit of nitrofurantoin is preferably 67.69 nM; and the detection limit of furazolidone is preferably 69.21 nM.

[0118] The invention provides a filter paper fluorescence sensor, comprising filter paper and a fluorescent material loaded on the surface of the filter paper, wherein the fluorescent material comprises the above-mentioned DA type organic fluorescent small molecule.

[0119] In the present invention, the preparation method of the filter paper fluorescence sensor preferably comprises the following steps:

[0120] The DA type organic fluorescent small molecule is dissolved in an organic solvent to obtain a solution containing the DA type organic fluorescent small molecule; the filter paper is immersed in the solution containing the DA type organic fluorescent small molecule and dried naturally to obtain the filter paper fluorescent sensor.

[0121] In the present invention, the organic solvent preferably includes one of acetone, tetrahydrofuran, anhydrous ethanol, N,N-dimethylformamide and dimethyl sulfoxide, and more preferably tetrahydrofuran.

[0122] In the present invention, the concentration of the solution containing DA-type organic fluorescent small molecules is preferably 0.5 to 2 μM, more preferably 1 μM. In the present invention, the immersion time is preferably 10 to 30 seconds.

[0123] The present invention provides a method for detecting nitrofuran antibiotics based on the filter paper fluorescence sensor, comprising the following steps:

[0124] The sample to be tested is added to the surface of the filter paper fluorescence sensor, the filter paper fluorescence sensor is irradiated with an ultraviolet light source, and the fluorescence color change of the filter paper fluorescence sensor is observed with the naked eye. If the fluorescence of the filter paper fluorescence sensor is completely quenched, it is determined that the sample to be tested contains nitrofuran antibiotics.

[0125] In the present invention, the nitrofuran antibiotics include one or more of nitrofurazone, nitrofurantoin and furazolidone.

[0126] In the present invention, the type and pretreatment method of the sample to be tested are the same as above and will not be described in detail. In the present invention, the applied volume of the sample to be tested is preferably 1 to 5 μL, more preferably 4 μL.

[0127] In the present invention, the ultraviolet light source is preferably a 365nm portable ultraviolet lamp.

[0128] The DA-type organic fluorescent small molecule, preparation method and application in detecting nitrofuran antibiotics provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0129] Example 1

[0130] The synthesis route of DA-type organic fluorescent small molecules is as follows:

[0131]

[0132] Under argon protection, 2-(benzothiazol-2-yl)-5-bromoaniline (184 mg, 0.6 mmol), 1-(4-phenylboronic acid pinacol ester)-1,2,2-triphenylethylene (330 mg, 0.72 mmol), tetrakis(triphenylphosphine)palladium (34 mg, 0.03 mmol), toluene (20 mL), potassium carbonate aqueous solution (2 mol / L, 12 mL) and ethanol (6 mL) were added to a 100 mL two-necked flask, heated at 90 ° C for 48 h; after the reaction was completed, it was cooled to room temperature, the reaction mixture was extracted with water and dichloromethane, the aqueous phase was discarded, the organic phase was collected, dried over anhydrous magnesium sulfate, and the organic solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified by column chromatography (dichloromethane-petroleum ether, volume ratio of 1:2), and recrystallized with dichloromethane and hexane to obtain a yellow-green solid product (200 mg, 60%), namely a DA-type organic fluorescent small molecule, denoted as P-BTTPE.

[0133] The NMR data of P-BTTPE prepared in this example are:

[0134] 1 H NMR (500MHz, CD 2 Cl 2)δ8.01(d,J=8.1Hz,1H),7.95(d,J=7.9Hz,1H),7.78(d,J=8.2Hz,1H),7.51(t,J=7.7Hz,1H),7.46(d,J=8.3Hz,2H),7.41(t,J=7.6Hz,1H),7.22-7.06(m,17H),7.04-6.97(m,2H),6.55(s,2H). Mass spectrum molecular ion peak: 556.593; theoretical molecular weight: 556.197.

[0135] Example 2

[0136] The synthesis route of DA-type organic fluorescent small molecules is as follows:

[0137]

[0138] Under argon protection, 2-(benzothiazol-2-yl)-5-bromoaniline (184 mg, 0.6 mmol), 4-(diphenylamino)phenylboronic acid (280 mg, 0.72 mmol), tetrakis(triphenylphosphine)palladium (34 mg, 0.03 mmol), toluene (12 mL), potassium carbonate aqueous solution (2 mol / L, 8 mL) and ethanol (4 mL) were added to a 100 mL double-necked bottle, heated at 90 ° C for 48 h; after the reaction, it was cooled to room temperature, the reaction mixture was extracted with water and dichloromethane, the aqueous phase was discarded, the organic phase was collected, dried with anhydrous magnesium sulfate, and the organic solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified by column chromatography (dichloromethane-petroleum ether, volume ratio of 1:2), and recrystallized from dichloromethane and hexane to obtain a yellow solid product (210 mg, 75%), i.e., a DA-type organic fluorescent small molecule, denoted as P-BTTPA.

[0139] The NMR data of P-BTTPA prepared in this example are:

[0140] 1 H NMR (500 MHz, DMSO) δ8.10 (d, J=7.8 Hz, 1H), 8.01 (d, J=8.1 Hz, 1H), 7.70 (d, J=8.3 Hz, 1H), 7.60 (d, J=8.6 Hz, 2H), 7.52 (t, J=7.6 Hz, 1H), 7.45-7.38 (m, 3H), 7.34 (t, J=7.8 Hz, 4H), 7.15 (s, 1H), 7.11-7.04 (m, 8H), 6.96 (d, J=8.3 Hz, 1H). Mass spectrum molecular ion peak: 469.000; theoretical molecular weight: 469.161.

[0141] Example 3

[0142] The synthesis route of DA-type organic fluorescent small molecules is as follows:

[0143]

[0144] Under argon protection, 2-(benzothiazol-2-yl)-5-bromoaniline (184 mg, 0.6 mmol), N-phenyl-3-carbazole boronic acid (208 mg, 0.72 mmol), tetrakis(triphenylphosphine)palladium (34 mg, 0.03 mmol), toluene (20 mL), potassium carbonate aqueous solution (2 mol / L, 12 mL) and ethanol (6 mL) were added to a 100 mL double-necked bottle, heated at 90 ° C for 48 h; after the reaction, it was cooled to room temperature, the reaction mixture was extracted with water and dichloromethane, the aqueous phase was discarded, the organic phase was collected, dried with anhydrous magnesium sulfate, and the organic solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified by column chromatography (dichloromethane-petroleum ether, volume ratio of 1:2), and recrystallized from dichloromethane and hexane to obtain a yellow solid product (230 mg, 82%), i.e., a DA-type organic fluorescent small molecule, denoted as P-BT3PCz.

[0145] The NMR data of P-BT3PCz prepared in this example are:

[0146] 1 H NMR (500MHz, DMSO) δ8.60(s,1H),8.38(d,J=7.7Hz,1H),8.11(d,J=7.8Hz,1H),8.03(d,J=8.0Hz,1H),7.77(d,J=8.3Hz,2H),7.74-7.67(m,4H ),7.58(t,J=7.2Hz,1H),7.53(t,J=7.6Hz,1H),7.47(dd,J=15.5,7.9Hz,4H),7.42(t,J=6.9Hz,2H),7.36-7.31(m,2H),7.13(d,J=8.3Hz,1H). 13 C NMR (151 MHz, DMSO-d 6)δ168.57,153.33,148.05,143.97,140.65,140.01,136.71,132.37,131.78,130.57,130.20,127.77,126.65,126.55,126.39,125.08,124.97,123.32,122.85,121.94,121.66,120.75,120.31,118.52,114.72,114.01,112.00,110.03,109.77. Mass spectrometry molecular ion peak: 467.573; Theoretical molecular weight: 467.146. Elemental analysis theoretical value: C 31 H 21 N 3 S: C, 79.63; H, 4.53; N, 8.99; actual value of elemental analysis: C, 83.39; H, 4.43; N, 9.05.

[0147] Example 4

[0148] The synthesis route of DA-type organic fluorescent small molecules is as follows:

[0149]

[0150] Under argon protection, 2-(benzothiazol-2-yl)-5-bromoaniline (184 mg, 0.6 mmol), 4-(9-carbazolyl)phenylboronic acid (208 mg, 0.72 mmol), tetrakis(triphenylphosphine)palladium (34 mg, 0.03 mmol), toluene (20 mL), potassium carbonate aqueous solution (2 mol / L, 12 mL) and ethanol (6 mL) were added to a 100 mL double-necked bottle, heated at 90 ° C for 48 h; after the reaction, it was cooled to room temperature, the reaction mixture was extracted with water and dichloromethane, the aqueous phase was discarded, the organic phase was collected, dried with anhydrous magnesium sulfate, and the organic solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified by column chromatography (dichloromethane-petroleum ether, volume ratio of 1:2), and recrystallized from dichloromethane and hexane to obtain a yellow solid product (189 mg, 67%), i.e., a DA-type organic fluorescent small molecule, denoted as P-BTPCz.

[0151] The NMR data of P-BTPCz prepared in this example are:

[0152] 1H NMR (500 MHz, DMSO) δ8.28 (d, J=7.8 Hz, 2H), 8.13 (d, J=7.9 Hz, 1H), 8.05 (d, J=8.0 Hz, 1H), 7.97 (d, J=8.3 Hz, 2H), 7.81 (d, J=8.2 Hz, 1H), 7.77 (d, J=8.3 Hz, 2H), 7.57-7.43 (m, 8H), 7.32 (t, J=7.2 Hz, 3H), 7.12 (d, J=8.3 Hz, 1H). Mass spectrum molecular ion peak: 466.923; theoretical molecular weight: 467.146.

[0153] Example 5

[0154] The synthesis route of DA-type organic fluorescent small molecules is as follows:

[0155]

[0156] Under argon protection, 2-(benzothiazol-2-yl)-5-bromoaniline (184 mg, 0.6 mmol), 9,9-dimethyl-10-phenyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9,10-dihydroacridine (296 mg, 0.72 mmol), tetrakis(triphenylphosphine)palladium (34 mg, 0.03 mmol), toluene (20 mL), potassium carbonate aqueous solution (2 mol / L, 12 mL) and ethanol (6 mL) were added to a 100 mL two-necked flask, heated at 90 ° C for 48 h under reflux; after the reaction was completed, the reaction mixture was cooled to room temperature, and the reaction mixture was extracted with water and dichloromethane, the aqueous phase was discarded, the organic phase was collected, dried over anhydrous magnesium sulfate, and the organic solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified by column chromatography (dichloromethane-petroleum ether, volume ratio of 1:2), and recrystallized with dichloromethane and hexane to obtain a yellow solid product (215 mg, 70%), which is a DA-type organic fluorescent small molecule, denoted as P-BTDMA.

[0157] The P-DMA NMR data prepared in this example are:

[0158] 1H NMR (500MHz, DMSO) δ8.08(d,J=7.9Hz,1H),8.00(d,J=8.0Hz,1H),7.78(d,J=1.5Hz,1H),7.73(t,J=7 .7Hz,2H),7.68(d,J=8.3Hz,1H),7.61(t,J=7.4Hz,1H),7.52(dd,J=17.5,7.9Hz,2H),7.45-7.35(m, 5H), 7.32(d, J=8.5Hz, 1H), 7.17(s, 1H), 7.00(t, J=7.3Hz, 1H), 6.94(t, J=8.3Hz, 2H), 6.23(d, J=8.6Hz, 1H), 6.16(d, J=8.0Hz, 1H), 1.72(s, 6H). Mass spectrum molecular ion peak: 494.280 (one methyl group is knocked off in the theoretical molecular weight); theoretical molecular weight: 509.193.

[0159] Example 6

[0160] Preparation of P-BT3PCz liquid phase fluorescence sensor:

[0161] The compound P-BT3PCz prepared in Example 3 was mixed with N,N-dimethylformamide to a concentration of 1×10 - 3 mol / L P-BT3PCz solution, take 3μL P-BT3PCz solution and place it in a quartz cuvette with 3mL of preparation solvent (the volume ratio of ultrapure water to N,N-dimethylformamide is 4:6) to obtain a concentration of 1×10 -6 mol / L P-BT3PCz liquid phase fluorescence sensor.

[0162] Example 7

[0163] Preparation of test systems containing real food samples (shrimp, chicken breast, lake water and tap water):

[0164] 2.0 g of chopped shrimp or chicken breast was added to 10 mL of N,N-dimethylformamide and ultrasonically extracted for 30 min. Then, the mixture was placed in a 50 mL centrifuge tube and centrifuged at 8000 rpm / min for 5 min. The supernatant was collected and filtered with a 0.45 μm organic membrane. The filtrate was used to prepare the nitrofuran antibiotic solution.

[0165] 30 mL of lake water or tap water samples were placed in 50 mL centrifuge tubes and centrifuged at 8000 rpm for 5 min. The supernatant of the pretreated water sample was filtered through a 0.45 μm water-based membrane and mixed with N,N-dimethylformamide in a ratio of 4:6 to prepare the P-BT3PCz liquid phase fluorescence sensor.

[0166] Example 8

[0167] Preparation of P-BT3PCz filter paper fluorescence sensor:

[0168] The compound P-BT3PCz prepared in Example 3 was mixed with tetrahydrofuran to prepare a concentration of 1×10 -6 mol / L P-BT3PCz solution. Immerse the filter paper in the P-BT3PCz solution for 10 seconds, take it out and dry it naturally to obtain the P-BT3PCz filter paper fluorescence sensor.

[0169] Example 9

[0170] Preparation of P-BTTPE, P-BTTPA, P-BTPCz and P-BTDMA liquid phase fluorescence sensors:

[0171] The compounds P-BTTPE, P-BTTPA, P-BTPCz and P-BTDMA prepared in Examples 1, 2, 4 and 5 were mixed with N,N-dimethylformamide to prepare a concentration of 1×10 -6 mol / L P-BTTPE, P-BTTPA, P-BTPCz and P-BTDMA solutions, and 3 mL of P-BTTPE, P-BTTPA, P-BTPCz and P-BTDMA solutions were placed in quartz cuvettes to obtain P-BTTPE, P-BTTPA, P-BTPCz and P-BTDMA liquid phase fluorescence sensors.

[0172] Application Example 1

[0173] Liquid phase detection of nitrofuran antibiotics using P-BT3PCz liquid phase fluorescence sensor:

[0174] The detection effect of the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 on nitrofuran antibiotics was verified.

[0175] 1) The absorption spectrum and emission spectrum of the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 were recorded using an ultraviolet absorption spectrometer and a fluorescence emission spectrometer, respectively. The results are as follows: Figure 1 As shown, the left curve represents the absorption spectrum and the right curve represents the emission spectrum.

[0176] Figure 1 It shows that the short-wavelength absorption of the P-BT3PCz liquid phase fluorescence sensor is mainly located at 302nm and 335nm, the long-wavelength absorption is mainly located at 392nm, and the fluorescence emission peak is located at 450nm.

[0177] 2) After gradually adding nitrofuran antibiotics (in the concentration range of 1 to 100 μM) to the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6, the changes in the fluorescence spectrum were observed. The measurement results were linearly fitted with the concentration of nitrofuran antibiotics (1 to 10 μM) as the abscissa and the fluorescence quenching rate at the corresponding concentration as the ordinate to obtain a standard curve. Figure 2 The fluorescence emission spectrum of the solution after adding furacilin to the P-BT3PCz liquid phase fluorescence sensor and the fluorescence quenching rate-concentration linear standard curve; Figure 2 In the figure, (a) is the fluorescence emission spectrum after adding different concentrations of furacilin (NFZ) to the P-BT3PCz liquid phase fluorescence sensor; (b) is the standard curve obtained by fitting the NFZ concentration and the fluorescence quenching rate obtained by the corresponding concentration.

[0178] Figure 3 The fluorescence emission spectrum of the solution after adding nitrofurantoin and the fluorescence quenching rate-concentration linear standard curve of the P-BT3PCz liquid phase fluorescence sensor; Figure 3 In the figure, (a) is the fluorescence emission spectrum after adding different concentrations of nitrofurantoin (NFT) to the P-BT3PCz liquid phase fluorescence sensor; (b) is the standard curve obtained by fitting the NFT concentration and the fluorescence quenching rate obtained by the corresponding concentration.

[0179] Figure 4 The fluorescence emission spectrum of the solution after adding furazolidone and the fluorescence quenching rate-concentration linear standard curve of the P-BT3PCz liquid phase fluorescence sensor; Figure 4 In the figure, (a) is the fluorescence emission spectrum after adding different concentrations of furazolidone (FZD) to the P-BT3PCz liquid phase fluorescence sensor; (b) is the standard curve obtained by fitting the FZD concentration and the fluorescence quenching rate obtained by the corresponding concentration.

[0180] like Figure 2 As shown in (a), with the increase of NFZ concentration, the emission peak of P-BT3PCz liquid phase fluorescence sensor at 450nm gradually weakened and the blue fluorescence intensity gradually decreased. Figure 2 (b) shows that the fitting curve has a good linear relationship in the range of 1 to 10 μM, and the calculated limit of detection (LOD) is 57.85 nM. Figure 3 As shown in (a), with the increase of NFT concentration, the emission peak of P-BT3PCz liquid phase fluorescence sensor at 450nm gradually weakened and the blue fluorescence intensity gradually decreased. Figure 3 (b) shows that the fitting curve has a good linear relationship in the range of 1 to 10 μM, and the calculated detection limit (LOD) is 63.45 nM. Figure 4As shown in (a), with the increase of FZD concentration, the emission peak of P-BT3PCz liquid phase fluorescence sensor at 450nm gradually weakened and the blue fluorescence intensity gradually decreased. Figure 4 (b) in the figure shows that the fitting curve has a good linear relationship in the range of 1 to 10 μM, and the calculated limit of detection (LOD) is 65.38 nM. These results show that the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 has a high sensitivity in detecting nitrofuran antibiotics.

[0181] 3) Add nitrofuran antibiotics (10 μM, 50 μM and 100 μM) to the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6, and observe and record the change of fluorescence intensity at 450 nm over time. The results are as follows: Figure 5 shown.

[0182] from Figure 5 As can be seen in (a), after adding NFZ (10μM, 50μM and 100μM) solution, the fluorescence intensity of the P-BT3PCz liquid phase fluorescence sensor at 450nm was rapidly quenched within 3s. Figure 5 (b) shows that after adding NFT (10μM, 50μM and 100μM) solution, the fluorescence intensity of P-BT3PCz liquid phase fluorescence sensor at 450nm was rapidly quenched within 3s. Figure 5 (c) in the figure shows that after adding FZD (10 μM, 50 μM and 100 μM) solution, the fluorescence intensity of the P-BT3PCz liquid phase fluorescence sensor at 450 nm was rapidly quenched within 3 seconds. These results show that the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 can almost instantaneously detect nitrofuran antibiotics and has an ultrafast response speed.

[0183] 4) Different interferences, including 14 common ions (Cu 2+ , Fe 3+ 、Al 3+ 、Zn 2+ 、Na + Mg 2+ , Ca 2+ 、Cd 2+ 、NO 3- 、NO 2- 、SO 4 2- , HSO 4- , HPO 4- ,I -) and 14 other types of antibiotics (metronidazole (MDZ), tinidazole (TDZ), enrofloxacin (ENR), norfloxacin (NFX), sulfamethoxazole (SMX), sulfadimethoxine (SMD), erythromycin (ERY), roxithromycin (ROX), tobramycin (TOB), streptomycin (STR), azithromycin (AZM), amoxicillin (AMX), thiamphenicol (THI) and florfenicol (FFC)) solutions were added to 28 portions of the P-BT3PCz liquid phase fluorescence sensor obtained in Example 6 (the concentration of each interferent was 100 μM), and the P-BT3PCz liquid phase sensor with nitrofuran antibiotics (100 μM) was used as a control. The color change of the obtained mixed solution under ultraviolet light at 365 nm was observed, and the change of fluorescence quenching rate was recorded and analyzed. The results are as follows: Figure 6 shown. Figure 6 In the figure, (a) is a bar graph of the fluorescence quenching rate after adding other different common ion interferents to the P-BT3PCz liquid phase fluorescence sensor; (b) is a bar graph of the fluorescence quenching rate after adding other different types of antibiotic interferents to the P-BT3PCz liquid phase fluorescence sensor.

[0184] from Figure 6 It can be seen that among all the test substances with the same concentration, only NFZ, NFT and FZD can cause the P-BT3PCz liquid phase fluorescence sensor to produce a significant fluorescence quenching response. These results show that the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 has excellent recognition specificity for the detection of nitrofuran antibiotics.

[0185] 5) Cu 2+ (100μM) / nitrofuran antibiotics (100μM), Fe 3+ (100μM) / nitrofuran antibiotics (100μM), Al 3+ (100μM) / nitrofuran antibiotics (100μM), Zn 2+ (100μM) / nitrofuran antibiotics (100μM), Na + (100μM) / nitrofuran antibiotics (100μM), Mg 2+ (100μM) / nitrofuran antibiotics (100μM), Ca 2+ (100μM) / nitrofuran antibiotics (100μM), Cd 2+ (100μM) / nitrofuran antibiotics (100μM), NO 3 - (100μM) / nitrofuran antibiotics (100μM), NO 2 -(100μM) / nitrofuran antibiotics (100μM), SO 4 2- (100 μM) / nitrofuran antibiotics (100 μM), HSO 4 - (100μM) / nitrofuran antibiotics (100μM), HPO 4 - (100μM) / nitrofuran antibiotics (100μM), I - (100μM) / Nitrofuran antibiotics (100μM), MDZ (100μM) / Nitrofuran antibiotics (100μM), TDZ (100μM) / Nitrofuran antibiotics (100μM), ENR (100μM) / Nitrofuran antibiotics (100μM), NFX (100μM) / Nitrofuran antibiotics (100μM), SMX (100μM) / Nitrofuran antibiotics (100μM), SMD (100μM) / Nitrofuran antibiotics (100μM), ERY (100μM) / Nitrofuran antibiotics (100μM), ROX (100μM) / Nitrofuran antibiotics (100μM), TOB (100 μM) / nitrofuran antibiotics (100 μM), STR (100 μM) / nitrofuran antibiotics (100 μM), AZM (100 μM) / nitrofuran antibiotics (100 μM), AMX (100 μM) / nitrofuran antibiotics (100 μM), THI (100 μM) / nitrofuran antibiotics (100 μM), and FFC (100 μM) / nitrofuran antibiotics (100 μM) solutions were added to 28 portions of the P-BT3PCz liquid phase fluorescence sensor obtained in Example 6, respectively. At the same time, the P-BT3PCz liquid phase fluorescence sensor added with nitrofuran antibiotics (100 μM) was used as a control, and the changes in the fluorescence quenching rate were recorded and analyzed. The results are shown in FIG. Figure 7 shown.

[0186] from Figure 7 It can be seen that the effect of the coexisting interferent on the fluorescence quenching rate is hardly noticeable compared with the case where only nitrofuran antibiotics are present. These results indicate that the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 has excellent anti-interference performance for the detection of nitrofuran antibiotics.

[0187] 6) Use an ultraviolet absorption spectrometer and a fluorescence emission spectrometer to respectively record the excitation spectrum and emission spectrum of the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 and the absorption spectrum of nitrofuran antibiotics, and record the fluorescence lifetime spectrum of the P-BT3PCz liquid phase fluorescence sensor after adding different concentrations of NFZ. Figure 8In the figure, (a) is the excitation spectrum and emission spectrum of the P-BT3PCz liquid phase fluorescence sensor and the absorption spectrum of the nitrofuran antibiotics, and (b) is the fluorescence lifetime spectrum of the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 after adding different concentrations of NFZ measured by a steady-state / transient spectrometer and the fluorescence lifetime obtained by fitting.

[0188] like Figure 8 As shown in (a), the excitation spectrum of the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 overlaps significantly with the absorption spectra of NFZ, NFT and FZD. When P-BT3PCz is excited by excitation light, nitrofuran antibiotics absorb the excitation light and show a fluorescence quenching response. Based on the spectral overlap, the fluorescence response mechanism is speculated to be the inner filter effect. Figure 8 The results in (b) show that the fluorescence lifetime of the mixed liquid after adding different concentrations of NFZ is basically unchanged compared with the fluorescence lifetime of P-BT3PCz when no NFZ is added. Therefore, it is determined that the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 detects nitrofuran antibiotics through the inner filter effect response mechanism.

[0189] In summary, the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 can detect nitrofuran antibiotics quickly, with high sensitivity, excellent selectivity and strong anti-interference ability through the inner filter effect. The excellent sensing performance shows that it has great potential for detecting nitrofuran antibiotics in actual environments.

[0190] Application Example 2

[0191] Detection of nitrofuran antibiotics in real food samples using P-BT3PCz liquid phase fluorescence sensor:

[0192] The preparation method described in Example 7 was used to verify the detection effect of the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 on NFZ, NFT and FZD in real food samples (shrimp, chicken breast, lake water and tap water).

[0193] 1) Add NFZ (2μM, 5μM, 10μM) solutions dissolved in various real food samples (shrimp, chicken breast, lake water and tap water) prepared according to Example 7 to the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6. Substitute the change results of the peak value in the obtained fluorescence spectrum into the standard curve obtained in Application Example 1 to deduce the concentration of NFZ measured by the P-BT3PCz liquid phase fluorescence sensor under this condition. The comparison results of the concentration of NFZ measured by the P-BT3PCz liquid phase fluorescence sensor prepared in Application Example 6 and the concentration of NFZ added to the actual standard are shown as follows: Fig.10 As shown in (a) in .

[0194] like Fig.10 As shown in (a), after adding NFZ (2μM, 5μM, 10μM) solutions dissolved in a variety of real food samples (shrimp, chicken breast, lake water and tap water), the P-BT3PCz liquid phase fluorescence sensor can also show fluorescence quenching corresponding to the concentration change. The concentration of NFZ measured by the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 is very close to the concentration result of NFZ added to the actual standard, with a recovery rate of 95.00% to 104.60%, and a relative standard deviation of less than 5.80% (n=3). This shows that the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 can accurately and quantitatively determine trace amounts of NFZ in real food samples.

[0195] 2) Add NFT (2μM, 5μM, 10μM) solutions dissolved in various real food samples (shrimp, chicken breast, lake water and tap water) prepared according to Example 7 to the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6. Substitute the change results of the peak value in the obtained fluorescence spectrum into the standard curve obtained in Application Example 1, and calculate the concentration of NFT measured by the P-BT3PCz liquid phase fluorescence sensor under this condition. The comparison results of the concentration of NFT measured by the P-BT3PCz liquid phase fluorescence sensor prepared in Application Example 6 and the concentration of NFT added to the actual standard are shown as follows: Fig.10 As shown in (b) in .

[0196] like Fig.10 As shown in (b), after adding NFT (2μM, 5μM, 10μM) solutions dissolved in a variety of real food samples (shrimp, chicken breast, lake water and tap water), the P-BT3PCz liquid phase fluorescence sensor can also show fluorescence quenching corresponding to the concentration change. The concentration of NFT measured by the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 is very close to the concentration result of the NFT added to the actual standard, with a recovery rate of 95.66% to 104.03%, and a relative standard deviation of less than 7.84% (n=3). This shows that the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 can accurately and quantitatively determine trace amounts of NFT in real food samples.

[0197] 3) Add the FZD (2μM, 5μM, 10μM) solutions dissolved in various real food samples (shrimp, chicken breast, lake water and tap water) prepared in accordance with Example 7 to the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6. Substitute the change results of the peak value in the obtained fluorescence spectrum into the standard curve obtained in Application Example 1 to estimate the concentration of FZD measured by the P-BT3PCz liquid phase fluorescence sensor under this condition. The comparison results of the concentration of FZD measured by the P-BT3PCz liquid phase fluorescence sensor prepared in Application Example 6 and the concentration of FZD added to the actual standard are shown as follows: Fig.10 As shown in (c) in .

[0198] like Fig.10 As shown in (c), after adding FZD (2μM, 5μM, 10μM) solutions dissolved in a variety of real food samples (shrimp, chicken breast, lake water and tap water), the P-BT3PCz liquid phase fluorescence sensor can also show fluorescence quenching corresponding to the concentration change. The concentration of FZD measured by the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 is very close to the concentration result of FZD added in the actual standard, with a recovery rate of 94.83% to 99.93%, and a relative standard deviation of less than 7.77% (n=3). This shows that the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 can accurately and quantitatively determine trace amounts of FZD in real food samples.

[0199] The above results show that the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 can accurately and quantitatively detect the residues of nitrofuran antibiotics in real meat samples and environmental water samples. Even at low concentrations, the detection results are not interfered by environmental samples, showing the versatility of the detection substrate, indicating that the P-BT3PCz liquid phase fluorescence sensor has the potential for application in real scenarios.

[0200] Application Example 3

[0201] P-BT3PCz liquid phase fluorescence sensor integrated with smartphone to build a quantitative evaluation system and its application in the detection of nitrofuran antibiotics in real food samples:

[0202] 1) In order to be free from the limitation of expensive instruments, the color perception error of the human eye is effectively eliminated by digital means. Different concentrations of NFZ are added to the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6, and the images of the P-BT3PCz liquid phase fluorescence sensor corresponding to the addition of different concentrations of NFZ under 365nm ultraviolet light are obtained using a smart phone. At the same time, the original image of the P-BT3PCz liquid phase fluorescence sensor is used as a control, and then the color analysis software in the mobile phone is used to output the RGB value of the fluorescence image in real time. The same method as above is applied to the determination of NFT and FZD. The determination results are linearly fitted with the concentration of nitrofuran antibiotics (1-10 μM) as the horizontal coordinate and the rate of change of the G value under 365nm ultraviolet light as the vertical coordinate to obtain a standard curve. Fig. 9 (a) is the fluorescence image of the P-BT3PCz liquid phase fluorescence sensor with different concentrations of NFZ added under 365nm ultraviolet light and its RGB analysis results; (b) is the standard curve obtained by fitting the G value change rate and the nitrofuran antibiotic concentration.

[0203] like Fig. 9As shown in (a), with the increase of NFZ concentration, the blue fluorescence intensity of P-BT3PCz liquid phase fluorescence sensor under 365nm ultraviolet light gradually decreases. Using the color analysis software in the mobile phone to output the RGB values ​​of each fluorescence image in real time, it can be observed that the R value and B value remain basically unchanged with the addition of nitrofuran antibiotics, but the G value gradually decreases with the increase of concentration. Fig. 9 The results in (b) show that the rate of change of the G value under 365nm ultraviolet light has a good linear relationship with the concentrations of NFZ, NFT and FZD (1-10μM), and the calculated LODs are 62.53nM, 67.69nM and 69.21nM, respectively. This shows that the image can be converted into RGB values ​​in real time by combining with a smartphone, and then substituted into the standard curve to obtain the corresponding nitrofuran antibiotic concentration, thereby achieving the purpose of visual quantitative detection of nitrofuran antibiotics. This method does not require the use of expensive instruments, and can effectively eliminate the human eye's perception error of color through digital means. It has the advantages of portability, speed and accuracy.

[0204] 2) Add NFZ (2μM, 5μM, 10μM) solutions dissolved in various real food samples (shrimp, chicken breast, lake water and tap water) prepared in accordance with Example 7 to the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6. The obtained fluorescence image is combined with a smartphone to perform RGB value analysis, and the result of the G value change rate is substituted into the standard curve obtained above to calculate the concentration of NFZ measured by the P-BT3PCz liquid phase fluorescence sensor under this condition. The comparison results of the NFZ concentration measured by the G value change rate and the concentration of NFZ added to the actual standard are shown as follows: Fig.10 As shown in (a) in .

[0205] like Fig.10 As shown in (a), after adding NFZ (2μM, 5μM, 10μM) solutions dissolved in a variety of real food samples (shrimp, chicken breast, lake water and tap water), the P-BT3PCz liquid phase fluorescence sensor can also show the corresponding concentration of G value changes. The concentration of NFZ measured by the G value change rate is very close to the concentration result of NFZ added to the actual standard, with a recovery rate of 97.63% to 104.53% and a relative standard deviation of less than 7.38% (n=3). This shows that the quantitative evaluation system constructed by integrating the P-BT3PCz liquid phase fluorescence sensor with a smartphone can be used to accurately quantify trace amounts of NFZ in real food samples.

[0206] 3) Add NFT (2μM, 5μM, 10μM) solutions dissolved in various real food samples (shrimp, chicken breast, lake water and tap water) prepared in accordance with Example 7 to the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6. The obtained fluorescence image is combined with a smartphone to perform RGB value analysis, and the result of the G value change rate is substituted into the standard curve obtained above to calculate the concentration of NFT measured by the P-BT3PCz liquid phase fluorescence sensor under this condition. The concentration of NFT measured by the G value change rate is compared with the concentration of NFT added to the actual standard as shown in the following figure. Fig.10 As shown in (b) in .

[0207] like Fig.10 As shown in (b), after adding NFT (2μM, 5μM, 10μM) solutions dissolved in a variety of real food samples (shrimp, chicken breast, lake water and tap water), the P-BT3PCz liquid phase fluorescence sensor can also show the corresponding concentration of G value changes. The concentration of NFT measured by the G value change rate is very close to the concentration result of the NFT added to the actual standard, with a recovery rate of 96.92% to 103.63%, and a relative standard deviation of less than 9.29% (n=3). This shows that the quantitative evaluation system constructed by integrating the P-BT3PCz liquid phase fluorescence sensor with a smartphone can be used to accurately quantify trace NFT in real food samples.

[0208] 4) Add the FZD (2μM, 5μM, 10μM) solution dissolved in various real food samples (shrimp, chicken breast, lake water and tap water) prepared in Example 7 to the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6. The obtained fluorescence image is combined with a smartphone to perform RGB value analysis, and the result of the G value change rate is substituted into the standard curve obtained above to calculate the concentration of FZD measured by the P-BT3PCz liquid phase fluorescence sensor under this condition. The comparison results of the FZD concentration measured by the G value change rate and the concentration of FZD added to the actual standard are shown as follows: Fig.10 As shown in (c) in .

[0209] like Fig.10 As shown in (c), after adding FZD (2μM, 5μM, 10μM) solutions dissolved in a variety of real food samples (shrimp, chicken breast, lake water and tap water), the P-BT3PCz liquid phase fluorescence sensor can also show the corresponding concentration of G value changes. The concentration of FZD measured by the G value change rate is very close to the concentration result of FZD added to the actual standard, with a recovery rate of 96.40% to 103.68% and a relative standard deviation of less than 5.54% (n=3). This shows that the quantitative evaluation system constructed by integrating the P-BT3PCz liquid phase fluorescence sensor with a smartphone can be used to accurately quantify trace amounts of FZD in real food samples.

[0210] In summary, the quantitative evaluation system constructed by integrating the P-BT3PCz liquid-phase fluorescence sensor with a smartphone can obtain the corresponding nitrofuran antibiotic concentration according to the rate of change of the G value, thereby achieving the purpose of visual quantitative detection of nitrofuran antibiotics. At the same time, it also performs well in the accurate quantitative detection of nitrofuran antibiotics in real meat samples and environmental water samples, showing the versatility of the detection substrate. These results show that the quantitative evaluation system provides a valuable technical reference for the highly sensitive, rapid and portable quantitative detection of nitrofuran antibiotics in complex environmental samples, and has broad application prospects.

[0211] Application Example 4

[0212] Qualitative detection of nitrofuran antibiotics by P-BT3PCz filter paper fluorescence sensor:

[0213] In order to qualitatively identify nitrofuran antibiotics in real time on site and increase their commercial value, 4 μL (100 μM) of nitrofurazone, nitrofurantoin and furazolidone were added to the P-BT3PCz filter paper fluorescence sensor prepared in Example 8. The fluorescence intensity change of the P-BT3PCz filter paper fluorescence sensor was observed under 365 nm ultraviolet light of a handheld ultraviolet analyzer. The results are as follows: Fig.11 shown. Fig.11 The filter paper strips shown in the figure are the original fluorescence intensity of the P-BT3PCz filter paper fluorescence sensor and the fluorescence intensity of the P-BT3PCz filter paper fluorescence sensor after adding nitrofurazone, nitrofurantoin or furazolidone.

[0214] like Fig.11 As shown, after adding 4 μL (100 μM) of nitrofurazone, nitrofurantoin and furazolidone, the fluorescence intensity of the P-BT3PCz filter paper fluorescence sensor was almost completely quenched under the 365 nm ultraviolet light of a handheld ultraviolet analyzer.

[0215] In summary, the P-BT3PCz filter paper fluorescence sensor prepared in the present invention can be used for on-site real-time qualitative identification of nitrofuran antibiotics by naked eyes without large-scale fluorescence testing instruments, and has the advantages of being light, portable, rapid, cheap, and environmentally friendly.

[0216] Application Example 5

[0217] Detection of nitrofuran antibiotics by liquid phase fluorescence sensors of P-BTTPE, P-BTTPA, P-BTPCz and P-BTDMA:

[0218] After adding nitrofurazone (100 μM), nitrofurantoin (100 μM) and furazolidone (100 μM) to the P-BTTPE, P-BTTPA, P-BTPCz and P-BTDMA liquid phase fluorescence sensors prepared in Example 9, the fluorescence spectra were observed. The results are as follows: Fig.12 , Fig.13 , Fig.14 and Fig.15 shown. Fig.12 The normalized fluorescence spectrum of P-BTTPE and the fluorescence spectrum after adding nitrofurazone, nitrofurantoin and furazolidone respectively. Fig.13 The normalized fluorescence spectrum of P-BTTPA and the fluorescence spectrum after adding nitrofurazone, nitrofurantoin and furazolidone respectively. Fig.14 The normalized fluorescence spectrum of P-BTPCz and the fluorescence spectrum after adding phosgene and DCP respectively. Fig.15 Normalized fluorescence spectrum of P-BTDMA and fluorescence spectrum after adding nitrofurazone, nitrofurantoin and furazolidone respectively.

[0219] like Fig.12 As shown in Figure 2, after adding nitrofurazone, nitrofurantoin and furazolidone, P-BTTPE showed that the original emission peak at 460 nm was almost completely quenched. Fig.13 As shown in Figure 2, after adding nitrofurazone, nitrofurantoin and furazolidone, P-BTTPA showed that the original emission peak at 468 nm was almost completely quenched. Fig.14 As shown in Figure 2, after adding nitrofurazone, nitrofurantoin and furazolidone, P-BTPCz showed that the original emission peak at 455nm was almost completely quenched. Fig.15 As shown in the figure, after adding nitrofurazone, nitrofurantoin and furazolidone, P-BTDMA showed that the original emission peak at 480nm was almost completely quenched. This shows that P-BTTPE, P-BTTPA, P-BTPCz and P-BTDMA liquid phase fluorescence sensors can achieve efficient fluorescence detection of nitrofurazone, nitrofurantoin and furazolidone.

[0220] In summary, the DA-type organic fluorescent small molecules provided by the present invention can realize the fluorescence detection of nitrofuran antibiotics. After the DA-type organic fluorescent small molecules provided by the present invention are prepared into fluorescent sensors, they can simultaneously realize the fluorescence detection of trace amounts of furazolidone, nitrofurantoin and furazolidone in the liquid phase, and have the advantages of fast response, high sensitivity, good selectivity, low detection cost, and strong anti-interference ability. Further, in order to overcome the inherent limitations of human visual perception, the RGB value of the fluorescent pattern is output in real time by combining the smartphone APP to realize the visualized quantitative detection of furazolidone, nitrofurantoin and furazolidone in real food samples. In addition, the DA-type organic fluorescent small molecules provided by the present invention can also be prepared into filter paper fluorescent sensors to achieve the purpose of rapid qualitative determination of furazolidone, nitrofurantoin and furazolidone.

[0221] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A DA-type organic fluorescent small molecule, characterized in that: It has the structure shown in formula I: In Formula I, R is 2. The DA type organic fluorescent small molecule according to claim 1, characterized in that: It has a structure shown in any one of Formula II to Formula VI:

3. The method for preparing the DA type organic fluorescent small molecule according to claim 1 or 2, characterized in that: The following steps are involved: A compound having a structure shown in formula A, a compound B, a catalyst, a solvent and an alkaline reagent are mixed and subjected to a Suzuki-Miyaura coupling reaction to obtain a DA-type organic fluorescent small molecule having a structure shown in formula I; In formula A, X is Cl, Br or I; The compound B has a structure shown in any one of formulas B1 to B11: In the structures represented by formulas B1 to B11, Y is a boronic acid group or a boronic ester group.

4. Use of the DA type organic fluorescent small molecule according to claim 1 or 2 or the DA type organic fluorescent small molecule prepared by the preparation method according to claim 3 in the detection of nitrofuran antibiotics.

5. The use according to claim 4, characterized in that: The nitrofuran antibiotics include one or more of nitrofurazone, nitrofurantoin and furazolidone; The nitrofuran antibiotics are nitrofuran antibiotics in food or water sources.

6. A liquid phase fluorescence sensor, characterized in that: The invention comprises a DA type organic fluorescent small molecule and a preparation solvent, wherein the DA type organic fluorescent small molecule is the DA type organic fluorescent small molecule according to claim 1 or 2 or the DA type organic fluorescent small molecule prepared by the preparation method according to claim 3.

7. A method for quantitative detection of nitrofuran antibiotics, characterized in that: The following steps are involved: Mixing the sample to be tested with the liquid phase fluorescence sensor according to claim 6 to obtain a fluorescence quenching rate; Obtaining the concentration of nitrofuran antibiotics in the sample to be tested according to the fluorescence quenching rate and a predetermined standard curve; The standard curve is a linear relationship curve between the concentration of nitrofuran antibiotics and the fluorescence quenching rate.

8. A visual quantitative detection method for nitrofuran antibiotics, characterized in that: The following steps are involved: Mixing the sample to be tested with the liquid phase fluorescence sensor according to claim 6, using a smart phone to obtain a fluorescence image of the resulting mixed solution and outputting the RGB value of the fluorescence image in real time to obtain the rate of change of the G value; The concentration of nitrofuran antibiotics in the sample to be tested is obtained according to the change rate of the G value and a predetermined standard curve.

9. A filter paper fluorescence sensor, characterized in that: The invention comprises filter paper and a fluorescent material loaded on the surface of the filter paper, wherein the fluorescent material comprises the DA type organic fluorescent small molecule according to claim 1 or 2 or the DA type organic fluorescent small molecule prepared by the preparation method according to claim 3.

10. The method for detecting nitrofuran antibiotics based on the filter paper fluorescence sensor according to claim 9, characterized in that: The following steps are involved: The sample to be tested is added to the surface of the filter paper fluorescence sensor, the filter paper fluorescence sensor is irradiated with an ultraviolet light source, and the fluorescence color change of the filter paper fluorescence sensor is observed with the naked eye. If the fluorescence of the filter paper fluorescence sensor is completely quenched, it is determined that the sample to be tested contains nitrofuran antibiotics.

Images