A d-a type organic fluorescent small molecule, a preparation method and application thereof in detection of nitrofurans antibiotics

Abstract

The application provides a D-A type organic fluorescent small molecule, a preparation method and application thereof in detection of nitrofuran antibiotics, and belongs to the technical field of fluorescent sensing. The D-A type organic fluorescent small molecule is composed of 2-(2-aminophenyl) benzothiazole and an electron-donating group, the excitation spectrum of the D-A type organic fluorescent small molecule is obviously overlapped with the absorption spectrum of nitrofuran antibiotics, fluorescence quenching response can be exhibited through internal filter effect, trace nitrofuran antibiotics can be detected in a liquid phase in a superfast, high-sensitivity, excellent selectivity and strong anti-interference manner. The application can be combined with a smart phone to build a quantitative evaluation system, real-time output of RGB values of a liquid-phase fluorescent sensor image can be realized, and rapid, accurate and visual quantitative detection of nitrofuran antibiotics in real food samples or water sources can be realized. In addition, the filter paper fluorescent sensor has the advantages of portability and simple preparation, and can rapidly identify nitrofuran antibiotics on site in an actual scene.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescent sensing technology, and in particular to a D-A type organic fluorescent small molecule, a preparation method and application thereof in detection of nitrofuran antibiotics. BACKGROUND

[0002] Veterinary drug residues caused by the abuse of antibiotics are a serious and widespread pollutant in food safety, which poses a great risk to human health and the environment. It is worth noting that nitrofuran antibiotics, as artificially synthesized broad-spectrum antibiotics, have been widely used in the prevention and treatment of diseases in poultry and aquaculture. Residual veterinary drugs can cause potential harm to various animals and plants and human health through the environment and food chain, leading to increased bacterial resistance in the body, causing allergic reactions or severe shock. Further studies have shown that the excessive accumulation of nitrofuran antibiotics can cause acute and chronic liver poisoning, prolonged blood clotting time, carcinogenesis, teratogenicity and other side effects. In view of the serious harm to health 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, under the drive of economic interests, the phenomenon of illegal abuse of nitrofuran antibiotics is still rampant. Therefore, it is essential to develop accurate and sensitive detection methods for nitrofuran antibiotic residues to protect the environment and human health.

[0003] At present, the main detection methods for 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 determination, time-resolved fluorescence immunochromatographic assay, 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 real-time and visual detection on site.

[0004] Based on the characteristics of fast response, high sensitivity and specificity, and easy integration into handheld devices, fluorescent sensors are considered more suitable for on-site applications. Currently, 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 unavoidable shortcomings, such as the difficulty in obtaining CDs materials with uniform particle size, the complexity of the purification process; MOFs, POPs and COFs have high synthesis difficulty and high cost of raw materials, and need to be ultrasonically pretreated when used.

[0005] In contrast, organic fluorescent small molecules have the advantages of simple synthesis, clear structure and good solubility, and are the ideal choice for nitrofuran antibiotic fluorescent probes. In addition, fluorescent sensors loaded on filter paper bases are also increasingly favored by people 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 not clear. Therefore, more cost-effective and efficient organic fluorescent small molecules need to be developed to detect nitrofuran antibiotic residues in actual environmental samples. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a D-A type organic fluorescent small molecule, a preparation method and its application in detecting nitrofuran antibiotics. The D-A type organic fluorescent small molecule provided by the present application can realize ultrafast, high-sensitivity and visual fluorescence detection of nitrofuran antibiotics.

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

[0008] The present application provides a D-A type organic fluorescent small molecule having the structure shown in formula I:

[0009]

[0010] In formula I, R is

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

[0012]

[0013] The present application provides a preparation method of the above-mentioned D-A type organic fluorescent small molecule, comprising the following steps:

[0014] Mixing a compound having the structure shown in formula A, compound B, a catalyst, a solvent and a basic reagent, performing Suzuki-Miyaura coupling reaction to obtain a D-A type organic fluorescent small molecule having the structure shown in formula I;

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

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

[0017]

[0018] In the structure shown in formula B1 to B11, Y is a boronic acid group or a boronic ester group.

[0019] The application provides application of the D-A type organic fluorescent small molecule in detection of nitrofuran antibiotics.

[0020] Preferably, the nitrofuran antibiotics include one or more of furacillin, furantoin and furazolidone.

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

[0022] The application provides a liquid-phase fluorescent sensor, which comprises a D-A type organic fluorescent small molecule and a preparation solvent, and the D-A type organic fluorescent small molecule is the D-A type organic fluorescent small molecule.

[0023] The application provides a quantitative detection method of nitrofuran antibiotics, which comprises the following steps.

[0024] The sample to be detected is mixed with the liquid-phase fluorescent sensor to obtain a fluorescence quenching rate.

[0025] The concentration of the nitrofuran antibiotics in the sample to be detected is obtained according to the fluorescence quenching rate and a predetermined standard curve.

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

[0027] The application provides a visual quantitative detection method of nitrofuran antibiotics, which comprises the following steps.

[0028] The sample to be detected is mixed with the liquid-phase fluorescent sensor, a fluorescence image of the obtained mixed solution is acquired by using a smart phone, and the RGB value of the fluorescence image is output in real time to obtain a change rate of the G value.

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

[0030] The application provides a filter paper fluorescent sensor, which comprises filter paper and a fluorescent material loaded on the surface of the filter paper, and the fluorescent material comprises the D-A type organic fluorescent small molecule.

[0031] The application provides a method for detecting nitrofuran antibiotics based on the filter paper fluorescent sensor, which comprises the following steps.

[0032] The sample to be detected is added to the surface of the filter paper fluorescent sensor, the filter paper fluorescent sensor is irradiated by using an ultraviolet light source, and the change of the fluorescence color of the filter paper fluorescent sensor is observed by naked eyes; if the fluorescence of the filter paper fluorescent sensor is completely quenched, it is determined that the sample to be detected contains nitrofuran antibiotics.

[0033] The application provides a D-A type organic fluorescent small molecule with a structure shown in formula I.The whole molecular skeleton of the D-A type organic fluorescent small molecule provided by the application is composed of an electron acceptor (2-(2-aminophenyl) benzothiazole) and an electron donor (i.e. the R group in formula I). The D-A type organic fluorescent small molecule material has the following beneficial effects:

[0034] (1) 2-(2-aminophenyl) benzothiazole is used as a main light-emitting building unit and an electron acceptor group, and after introducing an electron donor group such as tetraphenyl ethylene and triphenylamine, an intramolecular charge transfer state (ICT) can be formed, a D-A type organic fluorescent small molecule can be constructed, and the degree of ICT can be effectively adjusted to control the absorption wavelength and excitation wavelength.

[0035] (2) 2-(2-aminophenyl) benzothiazole has a rigid planar conjugated structure, and after being connected to an electron donor with a twisted configuration through a covalent bond, the interaction between molecules is reduced, high luminous efficiency is ensured, and efficient detection of nitrofuran antibiotics is facilitated.

[0036] (3) After the 2-(2-aminophenyl) benzothiazole group is connected to the electron donor group, the absorption peak at the long wavelength and the excitation spectrum of the group are red-shifted, the degree of overlap with the absorption peak of the nitrofuran antibiotic is increased, and the detection sensitivity and response time of the D-A type organic fluorescent small molecule to the nitrofuran antibiotic are further improved.

[0037] (4) In the D-A type organic fluorescent small molecule, P-BT3PCz can still maintain excellent optical properties in 40% water content, and can realize the fluorescent detection of nitrofuran antibiotics in a water and organic solvent mixed system. Meanwhile, 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 and the absorption spectrum of the nitrofuran antibiotic have obvious overlap, and the fluorescence lifetime does not change with the addition of the antibiotic, when the probe is excited by ultraviolet light, the excitation light is absorbed by the nitrofuran antibiotic, accompanied by rapid fluorescence quenching, realizing sensitive detection of nitrofuran antibiotics.

[0039] (6) The D-A type organic fluorescent small molecule is used as a guest material to prepare a filter paper fluorescent sensor. Since there is a certain torsion angle between the donor and the acceptor in the molecule, it is helpful to increase the molecular cavity, so that it can fully contact with the to-be-detected substance, thereby improving the detection performance.

[0040] This invention, based on the aforementioned DA-type organic fluorescent small molecules, prepares a liquid-phase fluorescence sensor and a filter paper fluorescence sensor for detecting nitrofuran antibiotics. These sensors enable ultrafast (3s), high-sensitivity (LOD below 57.85nM), excellent selectivity, and strong anti-interference capability (28 interfering substances) fluorescence detection of nitrofurans (NFZ), nitrofurantoin (NFT), and furazolidone (FZD). The liquid-phase fluorescence sensing system prepared in this invention can accurately and quantitatively detect nitrofuran antibiotics in real food samples and water sources (shrimp, chicken breast, lake water, tap water). Furthermore, this invention can be combined with a smartphone to construct a quantitative evaluation system, which can output the RGB values ​​of the fluorescence sensor image based on the aforementioned DA-type organic fluorescent small molecules in real time, achieving 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 portability and simple preparation, enabling rapid on-site identification of nitrofuran antibiotics in practical scenarios. Attached Figure Description

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

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

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

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

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

[0046] Figure 6 A bar chart showing the fluorescence quenching rate of the P-BT3PCz liquid phase fluorescence sensor after the addition of different interfering substances;

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

[0048] Figure 8The fluorescence lifetime spectra of the P-BT3PCz liquid-phase fluorescence sensor after adding different concentrations of NFZ;

[0049] Figure 9 The fluorescence image, RGB value analysis and standard curve of the P-BT3PCz liquid-phase fluorescence sensor after adding different concentrations of furazolidone;

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

[0051] Figure 11 The results of visual qualitative detection of nitrofuran antibiotics by the filter paper fluorescence sensor prepared based on P-BT3PCz;

[0052] Figure 12 The fluorescence emission spectra of the P-BTTPE liquid-phase fluorescence sensor after adding nitrofuran antibiotics respectively;

[0053] Figure 13 The fluorescence emission spectra of the P-BTTPA liquid-phase fluorescence sensor after adding nitrofuran antibiotics respectively;

[0054] Figure 14 The fluorescence emission spectra of the P-BTPCz liquid-phase fluorescence sensor after adding nitrofuran antibiotics respectively;

[0055] Figure 15 The fluorescence emission spectra of the P-BTDMA liquid-phase fluorescence sensor after adding nitrofuran antibiotics respectively. DETAILED DESCRIPTION

[0056] The application provides a D-A type organic fluorescent small molecule, which has the structure shown in formula I:

[0057]

[0058] In formula I, R is

[0059] In the application, the D-A type organic fluorescent small molecule has the structure shown in any one of formula II to formula VI:

[0060]

[0061] The D-A type organic fluorescent small molecule provided by the application has good solubility in an organic solvent, and is beneficial to preparation of a liquid-phase fluorescent sensor and a filter paper fluorescent sensor.

[0062] The application provides a preparation method of the D-A type organic fluorescent small molecule.

[0063] The compound with the structure shown in formula A, the compound B, a catalyst, a solvent and an alkaline reagent are mixed to perform a Suzuki-Miyaura coupling reaction, so that the D-A type organic fluorescent small molecule with the structure shown in formula I is obtained.

[0064] X in formula A is Cl, Br or I.

[0065] The compound B has the structure shown in any one of formula B1 to B11.

[0066]

[0067] Y in the structure shown in formula B1 to B11 is a boronic acid group or a boronic ester group.

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

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

[0070] In the application, the alkaline reagent preferably comprises one or both of potassium carbonate and potassium phosphate, and more preferably potassium carbonate; the alkaline reagent is preferably added in the form of an aqueous solution, and the concentration of the aqueous solution is preferably 1.5-2.5 mol / L, and more preferably 2 mol / L.

[0071] In the application, the molar ratio of the compound with the structure shown in formula A, the compound B, the catalyst and the alkaline reagent is preferably 1:(1-5):(0.04-0.1):(8-30), and more preferably 1:1.2:0.05:(10-20).

[0072] The application does not have special requirements for the solvent, and an organic solvent known to those skilled in the art can be used. As a specific embodiment of the application, the organic solvent is preferably a mixed solvent of toluene and ethanol, and the volume ratio of toluene to ethanol in the mixed solvent is preferably (2-5):1, and more preferably 3:1; the application does not have special requirements for the amount of the organic solvent, and the amount of the organic solvent can only be enough to completely dissolve the reaction raw materials.

[0073] In the application, the mixing mode is specifically preferably as follows:

[0074] (a) mixing a compound having the structure shown in formula A, compound B and a basic reagent to obtain a first mixed system;

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

[0076] (c) sequentially freezing and vacuumizing the second mixed system.

[0077] In the present application, the freezing in (b) and (c) is preferably liquid nitrogen freezing; the method of vacuumizing in (b) and (c) is not particularly limited in the present application, and any vacuumizing method known in the art can be used; the freezing and vacuumizing in (b) and (c) are performed once each time, and the operation is repeated, and the number of repetitions is preferably 3, the time of single freezing is preferably 10 min, and the time of single vacuumizing is preferably 5 min. The above feeding sequence and the pre-treatment of freezing and vacuumizing in the present application can remove oxygen in the reaction system as much as possible to avoid the oxidation and deactivation of the palladium catalyst, and provide favorable conditions for the Suzuki-Miyaura coupling reaction.

[0078] In the present application, the Suzuki-Miyaura coupling reaction is preferably performed under a protective atmosphere, and the protective atmosphere is preferably argon. In the present application, the temperature of the Suzuki-Miyaura coupling reaction is 85-90℃, and the time is preferably 24-48 h; as a specific embodiment of the present application, the temperature of the Suzuki-Miyaura coupling reaction can be 85℃, 88℃ or 90℃, and the time can be 24 h, 36 h or 48 h.

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

[0080] cooling the coupling reaction liquid to room temperature, extracting with water and dichloromethane, and collecting the organic phase;

[0081] sequentially drying and rotary evaporating the organic phase to obtain a crude product;

[0082] performing column chromatography purification on the crude product, and recrystallizing the obtained purified product to obtain the D-A type organic fluorescent small molecule purified product.

[0083] In the present application, the drying reagent used in the drying is preferably anhydrous magnesium sulfate; the rotary evaporation is used to remove the organic solvent. In the present application, the eluent used in the column chromatography is preferably dichloromethane and petroleum ether, and the volume ratio of the dichloromethane to the 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 as follows: after the purified product is just dissolved in a small amount of dichloromethane, hexane is added until the pure product is precipitated, that is, the D-A type organic fluorescent small molecule product.

[0084] The present application provides an application of the above-mentioned D-A type organic fluorescent small molecule in detecting nitrofuran antibiotics.

[0085] In the present application, the nitrofuran antibiotics include one or more of furacillin, furantoin and furazolidone.

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

[0087] The present application provides a liquid-phase fluorescent sensor comprising the above-mentioned D-A type organic fluorescent small molecule and a preparation solvent.

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

[0089] In the present application, the concentration of the D-A type organic fluorescent small molecule in the liquid-phase fluorescent sensor is preferably 0.5-2 μM, more preferably 1 μM. The present application does not have special requirements for the preparation method of the liquid-phase fluorescent sensor, and the D-A type organic fluorescent small molecule can be directly dissolved in the preparation solvent.

[0090] The present application provides a quantitative detection method of the above-mentioned nitrofuran antibiotic, comprising the following steps:

[0091] Mixing the sample to be tested with the above-mentioned liquid-phase fluorescent sensor to obtain a fluorescence quenching rate;

[0092] The concentration of the nitrofuran antibiotic in the sample to be detected is obtained according to the fluorescence quenching rate and a predetermined standard curve;

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

[0094] In the present application, the nitrofuran antibiotic includes one or more of furacillin, furantoin and furazolidone.

[0095] In the present application, the sample to be detected is preferably food or water source; the food is preferably animal source food. As a specific embodiment of the present application, the food is shrimp or chicken breast, and the water is lake water or tap water. In the present application, before detection, the sample to be detected is preferably pretreated, and when the sample to be detected is solid, the pretreatment preferably includes the following steps:

[0096] The crushed sample to be detected is mixed with an organic solvent, ultrasonic extraction and centrifugation are performed to obtain supernatant;

[0097] The supernatant is filtered.

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

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

[0100] In the present application, when the sample to be detected is liquid, the pretreatment preferably includes the following steps:

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

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

[0103] Mix the sample to be tested with the liquid-phase fluorescence sensor to obtain a fluorescence quenching rate. In the present application, the fluorescence quenching rate = 1-I / I0, wherein I0 is the initial fluorescence intensity of the organic small-molecule probe solution, and I is the fluorescence intensity of the solution after the addition of furacilin, nitrofurantoin or furazolidone.

[0104] The present application does not have special requirements for the detection of fluorescence intensity, and the fluorescence intensity can be detected by using the method well known to those skilled in the art. In the embodiments of the present application, the fluorescence intensity is preferably measured by using an RF-6000 fluorescence spectrophotometer, and the excitation wavelength is preferably determined according to the structure of the organic small-molecule fluorescent probe.

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

[0106] A nitrofuran antibiotic solution with a gradient known concentration is provided.

[0107] The sample to be tested is mixed with the liquid-phase fluorescence sensor, and the fluorescence image of the obtained mixture is acquired by using a smart phone, and the RGB value of the fluorescence image is output in real time to obtain the change rate of the G value.

[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-furacilin concentration standard curve, a fluorescence quenching rate-nitrofurantoin concentration standard curve, and a fluorescence quenching rate-furazolidone concentration standard curve.

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

[0110] The present application provides a visual quantitative detection method for nitrofuran antibiotics, which comprises the following steps:

[0111] Mix the sample to be tested with the liquid-phase fluorescence sensor, acquire the fluorescence image of the obtained mixture by using a smart phone, and output the RGB value of the fluorescence image in real time to obtain the change rate of the G value.

[0112] According to the change rate of the G value and a predetermined standard curve, the concentration of the nitrofuran antibiotic in the sample to be measured is obtained.

[0113] In the present application, the nitrofuran antibiotic includes one or more of nitrofurazone, furantoin and furazolidone.

[0114] In the present application, the kind of the sample to be measured and the pretreatment method are the same as above, and will not be repeated here.

[0115] In the present application, the change rate of the G value is equal to 1-G / G0, wherein G0 is the initial G value of the fluorescence pattern, and G is the G value of the fluorescence pattern after the sample to be measured is added.

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

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

[0118] The present application 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 D-A type organic fluorescent small molecule.

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

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

[0121] In the present application, 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 application, the concentration of the solution containing the D-A type organic fluorescent small molecule is preferably 0.5-2 μM, and more preferably 1 μM. In the present application, the soaking time is preferably 10-30 s.

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

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

[0125] In the present application, the nitrofuran antibiotics include one or more of furacillin, furantoin and furazolidone.

[0126] In the present application, the kind and pretreatment method of the sample to be detected are the same as above, and will not be repeated here. In the present application, the application volume of the sample to be detected is preferably 1-5 μL, and more preferably 4 μL.

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

[0128] The D-A type organic fluorescent small molecule, the preparation method and the application thereof in detecting nitrofuran antibiotics provided by the present application will be described in detail below in combination with examples, but they cannot be understood as limiting the protection scope of the present application.

[0129] Example 1

[0130] The synthesis of the D-A type organic fluorescent small molecule is as follows:

[0131]

[0132] Under the protection of argon, 2-(benzothiazole-2-yl)-5-bromo aniline (184 mg, 0.6 mmol), 1-(4-phenylboronic acid pinacol ester)-1,2,2-triphenyl ethylene (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 respectively added into a 100 mL two-necked flask, and the reaction was heated to reflux at 90°C for 48 h. After the reaction was completed, the reaction mixture was cooled to room temperature, extracted with water and dichloromethane, and the water phase was discarded. The organic phase was collected and 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 1:2), and recrystallized with dichloromethane and hexane to obtain a yellow-green solid product (200 mg, 60%), which is a D-A type organic fluorescent small molecule, recorded as P-BTTPE.

[0133] The nuclear magnetic resonance data of P-BTTPE prepared in the present example are as follows:

[0134] 1H NMR (500 MHz, CD2Cl2) δ 8.01 (d, J = 8.1 Hz, 1H), 7.95 (d, J = 7.9 Hz, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.46 (d, J = 8.3 Hz, 2H), 7.41 (t, J = 7.6 Hz, 1H), 7.22 - 7.06 (m, 17H), 7.04 - 6.97 (m, 2H), 6.55 (s, 2H). Mass spectrum molecular ion peak: 556.593; the theoretical molecular weight: 556.197.

[0135] Example 2

[0136] Synthesis of D-A type organic fluorescent small molecule, the synthetic route is as follows:

[0137]

[0138] Under argon protection, 2-(benzothiazol-2-yl)-5-bromo aniline (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 into a 100 mL two-necked flask respectively, heated to reflux at 90°C for 48 h; after the reaction was completed, cooled to room temperature, the reaction mixture was extracted with water and dichloromethane, the water phase was discarded, the organic phase was collected, dried with anhydrous magnesium sulfate, 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 1:2), recrystallized with dichloromethane and hexane to obtain a yellow solid product (210 mg, 75%), which was a D-A type organic fluorescent small molecule, recorded as P-BTTPA.

[0139] The P-BTTPA prepared in this example has the following nuclear magnetic resonance data:

[0140] 1 H NMR (500 MHz, DMSO) δ 8.10 (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; the theoretical molecular weight: 469.161.

[0141] Example 3

[0142] Synthesis of D-A type organic fluorescent small molecule, the synthetic route is as follows:

[0143]

[0144] Under argon protection, 2-(benzothiazol-2-yl)-5-bromo aniline (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), aqueous potassium carbonate solution (2 mol / L, 12 mL) and ethanol (6 mL) were added into a 100 mL two-necked flask respectively, and the reaction was heated to reflux at 90°C for 48 h. After the reaction was completed, the reaction mixture was cooled to room temperature, extracted with water and dichloromethane, and the water 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 1:2), and recrystallized with dichloromethane and hexane to obtain a yellow solid product (230 mg, 82%), which was a D-A type organic fluorescent small molecule, recorded as P-BT3PCz.

[0145] The P-BT3PCz prepared in this example has the following nuclear magnetic resonance data:

[0146] 1 H NMR (500 MHz, DMSO) δ 8.60 (s, 1H), 8.38 (d, J = 7.7 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 8.3 Hz, 2H), 7.74-7.67 (m, 4H), 7.58 (t, J = 7.2 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.47 (dd, J = 15.5, 7.9 Hz, 4H), 7.42 (t, J = 6.9 Hz, 2H), 7.36-7.31 (m, 2H), 7.13 (d, J = 8.3 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 168.57, 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 (Molecular ion peak): 467.573; Theoretical molecular weight: 467.146. Elemental analysis theoretical value: C 31 H 21 N3S: C, 79.63; H, 4.53; N, 8.99; Elemental analysis actual value: C, 83.39; H, 4.43; N, 9.05.

[0147] Example 4

[0148] Synthesis of D-A type organic fluorescent small molecule, the synthetic route is as follows:

[0149]

[0150] Under argon protection, 2-(benzothiazol-2-yl)-5-bromo aniline (184 mg, 0.6 mmol), 4-(9-carbazolyl) phenyl boronic acid (208 mg, 0.72 mmol), tetrakis (triphenylphosphine) palladium (34 mg, 0.03 mmol), toluene (20 mL), aqueous potassium carbonate solution (2 mol / L, 12 mL) and ethanol (6 mL) were added into a 100 mL two-port flask respectively, heated to reflux at 90°C for 48 h; after the reaction was completed, cooled to room temperature, the reaction mixture was extracted with water and dichloromethane, the water phase was discarded, the organic phase was collected, dried with anhydrous magnesium sulfate, 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 1:2), recrystallized with dichloromethane and hexane to obtain a yellow solid product (189 mg, 67%), which is a D-A type organic fluorescent small molecule, recorded as P-BTPCz.

[0151] The P-BTPCz prepared in this example has the following NMR data:

[0152] 1H NMR (500 MHz, DMSO) δ 8.28 (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 (m / e): 466.923; molecular weight: 467.146.

[0153] Example 5

[0154] Synthesis of D-A type organic fluorescent small molecule, the synthetic route is as follows:

[0155]

[0156] Under argon protection, 2-(benzothiazol-2-yl)-5-bromo aniline (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), aqueous potassium carbonate solution (2 mol / L, 12 mL) and ethanol (6 mL) were added into a 100 mL two-necked flask respectively, heated to reflux at 90°C for 48 h; after the reaction was completed, cooled to room temperature, the reaction mixture was extracted with water and dichloromethane, the water phase was discarded, the organic phase was collected, dried with anhydrous magnesium sulfate, 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 1:2), recrystallized with dichloromethane and hexane to obtain a yellow solid product (215 mg, 70%), which is a D-A type organic fluorescent small molecule, recorded as P-BTDMA.

[0157] The P-DMA prepared in this example has the following NMR data:

[0158] 1H NMR (500 MHz, DMSO) δ 8.08 (d, J = 7.9 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 1.5 Hz, 1H), 7.73 (t, J = 7.7 Hz, 2H), 7.68 (d, J = 8.3 Hz, 1H), 7.61 (t, J = 7.4 Hz, 1H), 7.52 (dd, J = 17.5, 7.9 Hz, 2H), 7.45 - 7.35 (m, 5H), 7.32 (d, J = 8.5 Hz, 1H), 7.17 (s, 1H), 7.00 (t, J = 7.3 Hz, 1H), 6.94 (t, J = 8.3 Hz, 2H), 6.23 (d, J = 8.6 Hz, 1H), 6.16 (d, J = 8.0 Hz, 1H), 1.72 (s, 6H). Mass (m / e) 494.280 (one methyl group lost from 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 prepare a P-BT3PCz solution with a concentration of 1 x 10 - 3 mol / L. 3 μL of the P-BT3PCz solution was placed in a quartz cuvette with 3 mL of the prepared solvent (ultra-pure water and N,N-dimethylformamide in a volume ratio of 4:6) to obtain a P-BT3PCz liquid-phase fluorescence sensor with a concentration of 1 x 10 -6 mol / L.

[0162] Example 7

[0163] Preparation of a detection system 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, and then placed in a 50 mL centrifuge tube and centrifuged at a speed of 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 a nitrofuran antibiotic solution.

[0165] 30 mL of lake water or tap water samples were placed in 50 mL centrifuge tubes and centrifuged at a speed of 8000 rpm for 5 min. The supernatant of the pretreated water sample was filtered with a 0.45 μm water-based membrane and mixed with N,N-dimethylformamide in a ratio of 4:6, and used to prepare a 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 P-BT3PCz solution with a concentration of 1 x 10 -6 mol / L. The filter paper was immersed in the P-BT3PCz solution for 10 s, and then taken out and naturally dried 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 respectively to prepare P-BTTPE, P-BTTPA, P-BTPCz and P-BTDMA solutions with a concentration of 1 x 10 -6 mol / L. 3 mL of the P-BTTPE, P-BTTPA, P-BTPCz and P-BTDMA solutions were respectively taken into a quartz cuvette 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 by 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 the emission spectrum of the P-BT3PCz liquid phase fluorescence sensor prepared in Example 6 were recorded by an ultraviolet absorption spectrometer and a fluorescence emission spectrometer respectively, and the results are shown in Figure 1 , wherein the left curve represents the absorption spectrum and the right curve represents the emission spectrum.

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

[0177] 2) The fluorescence spectra of P-BT3PCz solution were observed after adding nitrofuran antibiotics (concentration range from 1 to 100 μM) into the P-BT3PCz solution. The standard curve was obtained by linear fitting the concentration of nitrofuran antibiotics (1-10 μM) as the horizontal coordinate and the fluorescence quenching rate at the corresponding concentration as the vertical coordinate. Figure 2 The fluorescence emission spectra of P-BT3PCz solution and the linear standard curve of fluorescence quenching rate-concentration after adding nitrofurazone into the P-BT3PCz solution; Figure 2 In (a), the fluorescence emission spectra of P-BT3PCz solution after adding nitrofurazone (FZD) into the P-BT3PCz solution at different concentrations; in (b), the standard curve of FZD concentration and the fluorescence quenching rate at the corresponding concentration.

[0178] Figure 3 The fluorescence emission spectra of P-BT3PCz solution and the linear standard curve of fluorescence quenching rate-concentration after adding nitrofurantoin into the P-BT3PCz solution; Figure 3 In (a), the fluorescence emission spectra of P-BT3PCz solution after adding nitrofurantoin (NFT) into the P-BT3PCz solution at different concentrations; in (b), the standard curve of NFT concentration and the fluorescence quenching rate at the corresponding concentration.

[0179] Figure 4 The fluorescence emission spectra of P-BT3PCz solution and the linear standard curve of fluorescence quenching rate-concentration after adding nitrofurazone into the P-BT3PCz solution; Figure 4 In (a), the fluorescence emission spectra of P-BT3PCz solution after adding nitrofurazone (FZD) into the P-BT3PCz solution at different concentrations; in (b), the standard curve of FZD concentration and the fluorescence quenching rate at the corresponding concentration.

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

[0181] 3) Nitrofuran antibiotics (10 μM, 50 μM, and 100 μM) were added to the P-BT3PCz liquid-phase fluorescence sensor prepared in Example 6, and the change in fluorescence intensity at 450 nm over time was observed and recorded. The results are as follows: Figure 5 As shown.

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

[0183] 4) Different interfering substances, including 14 common ions (Cu... 2+ Fe 3+ Al 3+ Zn 2+ Na + Mg 2+ Ca 2+ Cd 2+ NO 3- NO 2- SO4 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 into 28 portions of the P-BT3PCz liquid-phase fluorescent sensor obtained in Example 6 (the concentration of each interferent was 100 μM), and the P-BT3PCz liquid-phase sensor to which nitrofuran antibiotics (100 μM) were added was used as a control. The color change of the obtained mixed solution under 365 nm ultraviolet light irradiation was observed, and the change in the fluorescence quenching rate was recorded and analyzed, and the results are shown in Figure 6 Figure 6 In the figure, (a) is a column chart of the fluorescence quenching rate after adding other different common ionic interferents to the P-BT3PCz liquid-phase fluorescent sensor; (b) is a column chart of the fluorescence quenching rate after adding other different other types of antibiotic interferents to the P-BT3PCz liquid-phase fluorescent sensor.

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

[0185] 5) Cu 2+ (100 μM) / nitrofuran antibiotics (100 μM), Fe 3+ (100 μM) / nitrofuran antibiotics (100 μM), Al 3+ (100 μM) / nitrofuran antibiotics (100 μM), Zn 2+ (100 μM) / nitrofuran antibiotics (100 μM), Na + (100 μM) / nitrofuran antibiotics (100 μM), Mg 2+ (100 μM) / nitrofuran antibiotics (100 μM), Ca 2+ (100 μM) / nitrofuran antibiotics (100 μM), Cd 2+ (100 μM) / nitrofuran antibiotics (100 μM), NO3 - (100 μM) / nitrofuran antibiotics (100 μM), NO2 - (100 μM) / nitrofuran antibiotics (100 μM), SO4 2- ​(100 μM) / nitrofuran antibiotics (100 μM), HSO4 - (100 μM) / nitrofuran antibiotics (100 μM), HPO4 - (100 μM) / nitrofuran antibiotics (100 μM), I - (100 μM) / nitrofuran antibiotics (100 μM), TDZ (100 μM) / nitrofuran antibiotics (100 μM), ENR (100 μM) / nitrofuran antibiotics (100 μM), NFX (100 μM) / nitrofuran antibiotics (100 μM), SMX (100 μM) / nitrofuran antibiotics (100 μM), SMD (100 μM) / nitrofuran antibiotics (100 μM), ERY (100 μM) / nitrofuran antibiotics (100 μM), ROX (100 μM) / nitrofuran antibiotics (100 μM), 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), FFC (100 μM) / nitrofuran antibiotics (100 μM) solutions were added into 28 portions of P-BT3PCz liquid phase fluorescent sensor prepared in Example 6, respectively, and the P-BT3PCz liquid phase fluorescent sensor with the addition of nitrofuran antibiotics (100 μM) was used as a control. The changes in fluorescence quenching rate were recorded and analyzed, and the results are shown in Table 2. Figure 7

[0186] From Figure 7 It can be seen that the influence of coexisting interferents on fluorescence quenching rate is hardly perceptible compared with the presence of only nitrofuran antibiotics. These results show that the P-BT3PCz liquid phase fluorescent sensor prepared in Example 6 has excellent anti-interference performance for the detection of nitrofuran antibiotics.

[0187] 6) The excitation spectrum and emission spectrum of the P-BT3PCz liquid phase fluorescent sensor prepared in Example 6 and the absorption spectrum of nitrofuran antibiotics were recorded by using a UV absorption spectrometer and a fluorescence emission spectrometer, respectively, and the fluorescence lifetime spectrum of the P-BT3PCz liquid phase fluorescent sensor after the addition of different concentrations of NFZ was recorded. Figure 8 ​In (a), the excitation and emission spectra of P-BT3PCz liquid-phase fluorescent sensor and the absorption spectra of nitrofuran antibiotics are shown. In (b), the fluorescence lifetime spectra of P-BT3PCz liquid-phase fluorescent sensor with different concentrations of NFZ are shown.

[0188] As shown in (a) of FIG. 6, the excitation spectrum of P-BT3PCz liquid-phase fluorescent sensor prepared in Example 6 overlaps with the absorption spectra of NFZ, NFT and FZD. When P-BT3PCz is excited by excitation light, nitrofuran antibiotics will absorb the excitation light, thereby showing a fluorescence quenching response. Based on the spectral overlap, the fluorescence response mechanism is speculated to be the inner filter effect. Figure 8 As shown in (b) of FIG. 6, the fluorescence lifetime of the mixed solution after adding different concentrations of NFZ is basically unchanged compared with the fluorescence lifetime of P-BT3PCz without NFZ. Therefore, it is determined that the P-BT3PCz liquid-phase fluorescent sensor prepared in Example 6 detects nitrofuran antibiotics through the inner filter effect response mechanism. Figure 8

[0189] In summary, the P-BT3PCz liquid-phase fluorescent sensor prepared in Example 6 can detect nitrofuran antibiotics through the inner filter effect with rapidity, high sensitivity, excellent selectivity and strong anti-interference ability. The excellent sensing performance indicates 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 by P-BT3PCz liquid-phase fluorescent sensor:

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

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

[0194] As shown in (a) of FIG. 7, the concentration of NFZ measured by the P-BT3PCz liquid-phase fluorescent sensor prepared in Example 6 is basically consistent with the actual standard concentration of NFZ added.​​Figure 10 As shown in (a), after adding NFZ solutions (2 μM, 5 μM, 10 μM) dissolved in various real food samples (shrimp, chicken breast, lake water, and tap water), the P-BT3PCz liquid chromometry sensor also showed fluorescence quenching corresponding to the concentration changes. The NFZ concentration measured by the P-BT3PCz liquid chromometry sensor prepared in Example 6 was very close to the concentration of NFZ added to the actual standard, with recoveries ranging from 95.00% to 104.60% and relative standard deviations less than 5.80% (n=3). This indicates that the P-BT3PCz liquid chromometry sensor prepared in Example 6 can accurately quantify trace amounts of NFZ in real food samples.

[0195] 2) Add NFT solutions (2 μM, 5 μM, 10 μM) prepared according to Example 7, dissolved in various real food samples (shrimp, chicken breast, lake water, and tap water), to the P-BT3PCz liquid chromometry sensor prepared in Example 6. Substitute the changes in peak values ​​in the obtained fluorescence spectra into the standard curve obtained in Example 1 to calculate the NFT concentration measured by the P-BT3PCz liquid chromometry sensor under these conditions. The comparison between the NFT concentration measured by the P-BT3PCz liquid chromometry sensor prepared in Example 6 and the concentration of NFT added to the actual standard is shown below. Figure 10 As shown in (b) of the diagram.

[0196] like Figure 10 As shown in (b), after adding NFT solutions (2 μM, 5 μM, 10 μM) dissolved in various real food samples (shrimp, chicken breast, lake water, and tap water), the P-BT3PCz liquid chromometry sensor also showed fluorescence quenching corresponding to the concentration changes. The NFT concentration measured by the P-BT3PCz liquid chromometry sensor prepared in Example 6 was very close to the concentration of NFT added to the actual standard, with recoveries ranging from 95.66% to 104.03% and relative standard deviations less than 7.84% (n=3). This indicates that the P-BT3PCz liquid chromometry sensor prepared in Example 6 can accurately quantify trace amounts of NFT in real food samples.

[0197] 3) Add FZD solutions (2 μM, 5 μM, 10 μM) prepared according to Example 7, dissolved in various real food samples (shrimp, chicken breast, lake water, and tap water), to the P-BT3PCz liquid-phase fluorescence sensor prepared in Example 6. Substitute the changes in peak values ​​in the obtained fluorescence spectra into the standard curve obtained in Example 1 to calculate the concentration of FZD measured by the P-BT3PCz liquid-phase fluorescence sensor under these conditions. The comparison between the concentration of FZD measured by the P-BT3PCz liquid-phase fluorescence sensor prepared in Example 6 and the concentration of FZD added to the actual standard is shown below.Figure 10 As shown in (c) in the figure.

[0198] like Figure 10 As shown in (c), after adding FZD solutions (2 μM, 5 μM, 10 μM) dissolved in various real food samples (shrimp, chicken breast, lake water, and tap water), the P-BT3PCz liquid chromatograph showed fluorescence quenching corresponding to the concentration changes. The FZD concentration measured by the P-BT3PCz liquid chromatograph prepared in Example 6 was very close to the concentration of FZD added to the actual standard, with a recovery rate of 94.83%–99.93% and a relative standard deviation of less than 7.77% (n=3). This indicates that the P-BT3PCz liquid chromatograph prepared in Example 6 can accurately quantify trace amounts of FZD in real food samples.

[0199] The above results demonstrate that the P-BT3PCz liquid chromatograph prepared in Example 6 can accurately and quantitatively detect nitrofuran antibiotic residues in real meat samples and environmental water samples. Even at low concentrations, the detection results are unaffected by environmental samples, exhibiting substrate versatility and indicating the potential of the P-BT3PCz liquid chromatograph for real-world applications.

[0200] Application Example 3

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

[0202] 1) To avoid the limitations of expensive instruments and effectively eliminate human visual color perception errors through digital means, the P-BT3PCz liquid fluorescence sensor prepared in Example 6 was added with different concentrations of NFZ. Images of the P-BT3PCz liquid fluorescence sensor under 365nm ultraviolet light with different concentrations of NFZ were acquired using a smartphone. The original images of the P-BT3PCz liquid fluorescence sensor were used as a control. The RGB values ​​of the fluorescence images were then output in real time using color analysis software on the smartphone. The same method was applied to the determination of NFT and FZD. The measurement results were linearly fitted with the concentration of nitrofuran antibiotics (1–10 μM) on the x-axis and the rate of change of G value under 365nm ultraviolet light on the y-axis to obtain a standard curve. Figure 9 (a) shows the fluorescence images and RGB analysis results of the P-BT3PCz liquid phase fluorescence sensor with different concentrations of NFZ added under 365nm ultraviolet light; (b) shows the standard curve obtained by fitting the change rate of G value with the concentration of nitrofuran antibiotics.

[0203] like Figure 9As shown in (a) of FIG. 6, with the increase of the concentration of NFZ, the blue fluorescence intensity of the P-BT3PCz liquid-phase fluorescent sensor gradually decreased under 365 nm ultraviolet light. Using the color analysis software in the mobile phone, the RGB values of each fluorescence image were output in real time, and it was observed that the R value and the B value were basically unchanged with the addition of nitrofuran antibiotics, but the G value gradually decreased with the increase of the concentration. Figure 9 As shown in (b) of FIG. 6, the results showed that the change rate of the G value had a good linear relationship with the concentration (1-10 μM) of NFZ, NFT and FZD under 365 nm ultraviolet light, and the LODs were calculated to be 62.53 nM, 67.69 nM and 69.21 nM, respectively. This indicates that the image can be converted into RGB values in real time by combining with the smart phone, and the corresponding concentration of nitrofuran antibiotics can be obtained by substituting the standard curve, so as to realize the purpose of visual quantitative detection of nitrofuran antibiotics. This method does not need to use expensive instruments, and can effectively eliminate the color perception error of the human eye by digital means, and has the advantages of portability, rapidness, accuracy, etc.

[0204] 2) The P-BT3PCz liquid-phase fluorescent sensor prepared in Example 6 was added with NFZ (2 μM, 5 μM, 10 μM) dissolved in various real food samples (shrimp, chicken breast, lake water and tap water) prepared according to Example 7. The fluorescence images obtained were analyzed by RGB values combined with a smart phone, and the change rate of the G value was brought into the standard curve obtained above to calculate the concentration of NFZ measured by the P-BT3PCz liquid-phase fluorescent sensor under this condition. The comparison results of the concentration of NFZ measured by the change rate of the G value and the actual standard concentration of NFZ added are shown in (a) of FIG. 7. Figure 10 As shown in (a) of FIG. 6.

[0205] As shown in (a) of FIG. 6. Figure 10 As shown in (a) of FIG. 6, after adding NFZ (2 μM, 5 μM, 10 μM) dissolved in various real food samples (shrimp, chicken breast, lake water and tap water), the P-BT3PCz liquid-phase fluorescent sensor also showed the change of the G value corresponding to the concentration. The concentration of NFZ measured by the change rate of the G value was very close to the actual standard concentration of NFZ added, and the recovery rate reached 97.63%-104.53%, and the relative standard deviation was less than 7.38% (n=3). This indicates that the P-BT3PCz liquid-phase fluorescent sensor integrated with the smart phone can be used for accurate quantitative determination of trace NFZ in real food samples.

[0206] 3) Add NFT solutions (2 μM, 5 μM, 10 μM) prepared according to Example 7, dissolved in various real food samples (shrimp, chicken breast, lake water, and tap water), to the P-BT3PCz liquid chromatograph prepared in Example 6. Analyze the obtained fluorescence images using a smartphone to obtain RGB values. Substitute the G-value change rate results into the obtained standard curve to calculate the NFT concentration measured by the P-BT3PCz liquid chromatograph under these conditions. The comparison between the NFT concentration measured using the G-value change rate and the NFT concentration added to the actual standard is shown below. Figure 10 As shown in (b) of the diagram.

[0207] like Figure 10 As shown in (b), after adding NFT solutions (2 μM, 5 μM, 10 μM) dissolved in various real food samples (shrimp, chicken breast, lake water, and tap water), the P-BT3PCz liquid chromatography-fluorescence sensor also showed changes in the corresponding concentrations of G values. The NFT concentrations measured using the rate of change of G values ​​were very close to the concentrations of NFTs added to the actual standards, with recoveries ranging from 96.92% to 103.63% and relative standard deviations less than 9.29% (n=3). This indicates that the P-BT3PCz liquid chromatography-fluorescence sensor, integrated with a smartphone to construct a quantitative assessment system, can be used to accurately quantify trace amounts of NFTs in real food samples.

[0208] 4) Add FZD solutions (2 μM, 5 μM, 10 μM) prepared according to Example 7, dissolved in various real food samples (shrimp, chicken breast, lake water, and tap water), to the P-BT3PCz liquid-phase fluorescence sensor prepared in Example 6. Perform RGB value analysis on the obtained fluorescence images using a smartphone. Substitute the result of the G-value change rate into the obtained standard curve to calculate the FZD concentration measured by the P-BT3PCz liquid-phase fluorescence sensor under these conditions. The comparison between the FZD concentration measured using the G-value change rate and the concentration of FZD added to the actual standard is shown below. Figure 10 As shown in (c) in the figure.

[0209] like Figure 11 As shown in (c), after adding FZD solutions (2 μM, 5 μM, 10 μM) dissolved in various real food samples (shrimp, chicken breast, lake water, and tap water), the P-BT3PCz liquid chromatography-fluorescence sensor also showed changes in the corresponding concentrations of G values. The FZD concentration measured using the rate of change of G values ​​was very close to the concentration of FZD added to the actual standard, with recoveries ranging from 96.40% to 103.68% and relative standard deviations less than 5.54% (n=3). This indicates that the P-BT3PCz liquid chromatography-fluorescence sensor, integrated with a smartphone to construct a quantitative assessment system, can be used to accurately quantify trace amounts of FZD in real food samples.

[0210] In summary, the P-BT3PCz liquid-phase fluorescent sensor and the quantitative evaluation system integrated with a smartphone can obtain the corresponding concentration of nitrofuran antibiotics according to the change rate of G value, thereby realizing the purpose of visual quantitative detection of nitrofuran antibiotics. At the same time, it also performs well in accurately detecting nitrofuran antibiotics in real meat samples and environmental water samples, showing the universality of the detection substrate. These results show that the quantitative evaluation system provides a valuable technical reference for the high-sensitivity, rapid, 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 fluorescent sensor:

[0213] In order to be able to qualitatively identify nitrofuran antibiotics in real time on site and increase its commercial value, the P-BT3PCz filter paper fluorescent sensor prepared in Example 8 was added with 4 μL (100 μM) of furacillin, furantoin and furazolidone, respectively. Under the 365 nm ultraviolet light of the handheld ultraviolet analyzer, the change of the fluorescence intensity of the P-BT3PCz filter paper fluorescent sensor was observed, and the results are shown in Figure 11 . Figure 11 The filter paper strips shown in the figure are the original fluorescence intensity of the P-BT3PCz filter paper fluorescent sensor, the fluorescence intensity of the P-BT3PCz filter paper fluorescent sensor after adding furacillin, furantoin or furazolidone.

[0214] As shown in Figure 12 , after adding 4 μL (100 μM) of furacillin, furantoin and furazolidone, the fluorescence intensity of the P-BT3PCz filter paper fluorescent sensor under the 365 nm ultraviolet light of the handheld ultraviolet analyzer was almost completely quenched.

[0215] In summary, the P-BT3PCz filter paper fluorescent sensor prepared by the present application can qualitatively identify nitrofuran antibiotics in real time on site by naked eye without large-scale fluorescent testing instruments, and has the advantages of lightness, portability, rapidness, low cost, environmental friendliness and the like.

[0216] Application Example 5

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

[0218] The fluorescence spectra of the P-BTTPE, P-BTTPA, P-BTPCz and P-BTDMA liquid-phase fluorescent sensors prepared in Example 9 were observed after adding furacilin (100 μM), furantoin (100 μM) and furazolidone (100 μM) respectively, and the results are shown in Figure 13 , Figure 14 , Figure 15 and Figure 12 . Figure 13 is the normalized fluorescence spectrum of P-BTTPE and the fluorescence spectra after adding furacilin, furantoin and furazolidone respectively. Figure 14 is the normalized fluorescence spectrum of P-BTTPA and the fluorescence spectra after adding furacilin, furantoin and furazolidone respectively. Figure 15 is the normalized fluorescence spectrum of P-BTPCz and the fluorescence spectra after adding phosgene and DCP respectively. Figure 12 is the normalized fluorescence spectrum of P-BTDMA and the fluorescence spectra after adding furacilin, furantoin and furazolidone respectively.

[0219] As shown in Figure 13 , P-BTTPE showed almost complete quenching of the original emission peak at 460 nm after adding furacilin, furantoin and furazolidone. As shown in Figure 14 , P-BTTPA showed almost complete quenching of the original emission peak at 468 nm after adding furacilin, furantoin and furazolidone. As shown in Figure 15 , P-BTPCz showed almost complete quenching of the original emission peak at 455 nm after adding furacilin, furantoin and furazolidone. As shown in ​ , P-BTDMA showed almost complete quenching of the original emission peak at 480 nm after adding furacilin, furantoin and furazolidone. This shows that the P-BTTPE, P-BTTPA, P-BTPCz and P-BTDMA liquid-phase fluorescent sensors can all achieve efficient fluorescence detection of furacilin, furantoin and furazolidone.

[0220] In summary, the D-A type organic fluorescent small molecule provided by the present application can realize the fluorescent detection of nitrofurans antibiotics. After the D-A type organic fluorescent small molecule provided by the present application is prepared into a fluorescent sensor, it can simultaneously realize the fluorescent detection of trace furacin, nitrofurantoin and furazolidone in a liquid phase, and has the advantages of rapid response, high sensitivity, good selectivity, low detection cost, strong anti-interference ability and the like. Further, in order to overcome the inherent limitations of human visual perception, the RGB values of the fluorescent pattern output in real time are combined with the intelligent mobile phone APP, so that the visual quantitative detection of furacin, nitrofurantoin and furazolidone in real food samples is realized. In addition, the D-A type organic fluorescent small molecule provided by the present application can also be prepared into a filter paper fluorescent sensor, so as to realize the purpose of rapid qualitative determination of furacin, nitrofurantoin and furazolidone.

[0221] The above only describes the preferred embodiments of the present application, and it should be pointed out that, for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A D-A type organic fluorescent small molecule, characterized in that, A compound having a structure shown in formula I: In formula I, R is 2.The method for preparing the D-A type organic fluorescent small molecule of claim 1, characterized in that, The method comprises the following steps: mixing a compound having a structure shown in formula A, a compound B, a catalyst, a solvent and a basic reagent to perform a Suzuki-Miyaura coupling reaction, so as to obtain a D-A type organic fluorescent small molecule having a structure shown in formula I; X in formula A is Cl, Br or I; The compound B has a structure shown in any one of formula B1 to B5: In the structure shown in formula B1 to B5, Y is a boronic acid group or a boronic ester group.

3. Application of the D-A type organic fluorescent small molecule in claim 1 in detection of nitrofuran antibiotics. The nitrofuran antibiotics are selected from one or more of furacillin, furaltadone and furazolidone. The nitrofuran antibiotics are nitrofuran antibiotics in food or water sources.

4. A liquid phase fluorescent sensor, characterized by, The method comprises the following steps:

5. A method for quantitative detection of nitrofuran antibiotics for non-disease diagnosis and therapeutic purposes, characterized by, mixing a compound having a structure shown in formula A, a compound B, a catalyst, a solvent and a basic reagent to perform a Suzuki-Miyaura coupling reaction, so as to obtain a D-A type organic fluorescent small molecule having a structure shown in formula I; The method comprises the following steps: mixing the sample to be detected with the liquid-phase fluorescent sensor in claim 4 to obtain a fluorescence quenching rate; obtaining the concentration of the nitrofuran antibiotics in the sample to be detected according to the fluorescence quenching rate and a predetermined standard curve; The standard curve is a linear relationship curve of the concentration of the nitrofuran antibiotics and the fluorescence quenching rate.

6. A method for visualizing and quantifying nitrofuran antibiotics for non-disease diagnostic therapeutic purposes, characterized in that, The nitrofuran antibiotics are selected from one or more of furacillin, furaltadone and furazolidone. The method comprises the following steps: mixing the sample to be detected with the liquid-phase fluorescent sensor in claim 4, using a smart phone to acquire a fluorescence image of the obtained mixed solution and outputting RGB values of the fluorescence image in real time, and obtaining a change rate of G value; obtaining the concentration of the nitrofuran antibiotics in the sample to be detected according to the change rate of G value and a predetermined standard curve; 7. A filter paper fluorescence sensor characterized in that, The nitrofuran antibiotics are selected from one or more of furacillin, furaltadone and furazolidone.

8. A method for detecting nitrofuran antibiotics for non-disease diagnosis and therapeutic purposes, characterized by, The method comprises the following steps: mixing the sample to be detected with the liquid-phase fluorescent sensor in claim 4, using a smart phone to acquire a fluorescence image of the obtained mixed solution and outputting RGB values of the fluorescence image in real time, and obtaining a change rate of G value; obtaining the concentration of the nitrofuran antibiotics in the sample to be detected according to the change rate of G value and a predetermined standard curve; The nitrofuran antibiotics are selected from one or more of furacillin, furaltadone and furazolidone. The method comprises the following steps: mixing the sample to be detected with the liquid-phase fluorescent sensor in claim 4, using a smart phone to acquire a fluorescence image of the obtained mixed solution and outputting RGB values of the fluorescence image in real time, and obtaining a change rate of G value; obtaining the concentration of the nitrofuran antibiotics in the sample to be detected according to the change rate of G value and a predetermined standard curve; The nitrofuran antibiotics are selected from one or more of furacillin, furaltadone and furazolidone.

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