Ratio fluorescent probe for detecting aureomycin in meat food and detection method

Abstract

The invention provides a ratio fluorescent probe for detecting aureomycin in meat food and a detection method. After an aureomycin solution is added into the prepared Au NCs ratiometric fluorescent probe solution, a fluorescence emission band of a reaction system at 620 nm is almost unchanged, and a fluorescence emission band at 425 nm is remarkably enhanced. Meanwhile, the color of a reaction system under an ultraviolet lamp is gradually changed from red to purple along with the increase of the concentration of the aureomycin, and quantitative detection of the aureomycin can be realized by utilizing the characteristics. The detection method disclosed by the invention comprises two detection methods, one is a ratio fluorescent probe detection method, and quantitative detection is realized by utilizing linear correlation between the F425 / F620 fluorescence intensity ratio and the chlortetracycline concentration. The other method is a visual quantitative detection method, a fluorescent paper chip image is collected by using a smart phone and converted into a corresponding RGB value, and quantitative detection is realized by using a linear relationship between an R / B value and the chlortetracycline concentration. The detection method disclosed by the invention has the advantages of high selectivity, high sensitivity, simplicity in operation, obvious phenomenon and good stability and repeatability.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence detection, and particularly relates to a ratio fluorescence probe for detecting chlortetracycline in meat foods and a detection method therefor. Background Art

[0002] Chlortetracycline (CTC) is a tetracycline antibiotic drug with strong antibacterial and anti-inflammatory effects. Clinically, it is mainly used to treat infectious diseases caused by bacteria, etc. However, it has relatively large toxic and side effects, and improper use is likely to cause side effects such as gastrointestinal disorders and hepatotoxicity. In addition, CTC is often used as a feed additive, which can promote animal growth, improve feeding efficiency, and at the same time has the effects of preventing animal diseases, improving digestion and metabolism, and inhibiting harmful microorganisms. However, excessive use will lead to the residue of CTC in agricultural and sideline products, which will seriously threaten human health, cause adverse physiological reactions such as allergic reactions, and also cause environmental pollution and bacterial drug resistance. Currently, the commonly used determination methods for CTC include high performance liquid chromatography, immunochromatography, chemiluminescence method, and microbiological method, etc. However, these methods are cumbersome to operate and are easily affected by impurities, and the anti-interference effect is poor.

[0003] In recent years, metal nanoclusters (MNCs) have shown great potential in the fields of fluorescence nanoprobes and bioimaging due to their advantages such as ultra-small size, photostability, low toxicity, biocompatibility, tunable emission, and large Stokes shift. Currently, proteins, polypeptides, and DNA are used as effective reducing agents and biological templates to synthesize MNCs. Some researchers have proposed some ratio fluorescence methods for detecting CTC using MNCs as probes, but there are problems such as high detection method cost, low sensitivity, poor selectivity, and complex operation. Therefore, it is of great significance to develop a more simple and rapid CTC quantitative detection method. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a ratio fluorescence probe for detecting chlortetracycline in meat foods for quantitative detection of chlortetracycline in meat foods.

[0005] This purpose of the present invention is achieved by the following technical solutions: A ratio fluorescence probe for detecting chlortetracycline in meat foods is provided, and the preparation method includes the following steps:

[0006] (1-1) Mix an aqueous solution of BSA and AuHCl 4 and stir vigorously, add a NaOH solution, and oscillate at a constant temperature to obtain a light yellow transparent solution;

[0007] (1-2) Purify the above transparent solution using a dialysis membrane to obtain an Au NCs gold nanocluster solution emitting red fluorescence;

[0008] (1-3) The above Au NCs gold nanocluster solution is dried to obtain the finished Au NCs gold nanocluster product, which is stored for later use;

[0009] (1-4) The above Au NCs gold nanocluster product is formulated into an aqueous solution, and a buffer solution is added to obtain a ratio fluorescence probe;

[0010] In step (1-4), the concentration of the ratio fluorescence probe is 10 μg / mL, and the pH of the buffer solution is 10.

[0011] Furthermore, in step (1-1), the mass and volume ratios of BSA, AuHCl 4 , and NaOH are 500 mg∶10 mL∶1 mL. The vigorous stirring time is 2 minutes, and the constant temperature oscillation condition is oscillation at 37 °C for 12 h.

[0012] Furthermore, in step (1-2), the dialysis membrane purification condition is purification with a 1000 Da dialysis membrane for 12 h.

[0013] Furthermore, in step (1-4), the buffer solution is Tris-HCl.

[0014] After adding the chlortetracycline solution to the above ratio fluorescence probe solution, the fluorescence emission band of the system at 620 nm hardly changes, while the fluorescence emission band at 425 nm is significantly enhanced; at the same time, with the change of the chlortetracycline solution concentration, the color of the system under ultraviolet light also changes.

[0015] The second object of the present invention is to provide a method for detecting chlortetracycline in meat food.

[0016] This object of the present invention is achieved by the following technical solution. A method for detecting chlortetracycline in meat food using the above ratio fluorescence probe is provided, including the following steps:

[0017] (2-1) Add the test solution to the ratio fluorescence probe solution and incubate at 10 °C to 50 °C for 5 to 10 min;

[0018] (2-2) Detect at an excitation wavelength of 350 nm, record the fluorescence emission spectrum in the range of 370 to 670 nm, and obtain the fluorescence intensity ratio at 425 nm and 620 nm, that is, F 425 / F 620 ;

[0019] (2-3) Calculate the concentration of chlortetracycline in the test solution through the standard curve.

[0020] Furthermore, in step (2-1), the incubation conditions are preferably 30 °C and 5 min.

[0021] Further, the standard curve in step (2-3) is: F 425 / F 620 = 0.1718[CTC] + 1.2874(R 2 = 0.9987), where [CTC] represents the concentration of chlortetracycline.

[0022] The third object of the present invention is to provide another method for detecting chlortetracycline in meat food.

[0023] This object of the present invention is achieved by the following technical solution: providing a method for detecting chlortetracycline in meat food using the above-mentioned ratio fluorescence probe, comprising the following steps:

[0024] (3-1) Prepare a fluorescent paper chip: The filter paper is soaked in the ratio fluorescence probe solution, taken out, and dried for standby.

[0025] (3-2) Immerse the fluorescent paper chip prepared in step (3-1) into the test solution and incubate for 1 min;

[0026] (3-3) Air-dry the fluorescent paper chip after incubation in step (3-2), collect the image of the fluorescent paper chip under a 365 nm ultraviolet lamp using software, perform color recognition on the collected image, convert it into the corresponding RGB value, and calculate the R / B value;

[0027] (3-4) Calculate the concentration of chlortetracycline in the test solution through the standard curve.

[0028] Further, the time for the filter paper to be immersed in the ratio fluorescence probe in step (3-1) is 30 min.

[0029] Further, the standard curve in step (3-4) is: R / B = 0.0255[CTC] + 1.4807(R 2 = 0.9847), where [CTC] represents the concentration of chlortetracycline.

[0030] Advantages of the present invention:

[0031] (1) The ratio fluorescence probe constructed in the present invention can achieve quantitative detection of chlortetracycline in meat food. This probe system has good selectivity, sensitivity, and excellent repeatability. At an excitation wavelength of 350 nm, the fluorescence ratio F 425 / F 620 has a linear relationship with the concentration of CTC in the range of 0.3 - 30 μmol / L, and the linear equation is F 425 / F 620 = 0.1718[CTC] + 1.2874(R 2 = 0.9987), and the lowest detection limit is 0.18 μmol / L.

[0032] (2) After the chlortetracycline in the solution to be measured reacts with the ratio fluorescence probe, the color of the system under ultraviolet light gradually changes from red to purple as the concentration of chlortetracycline increases, and the color change is obvious. On this basis, software is used to perform color recognition on the collected images and convert them into corresponding RGB values. The ratio of R / B to the CTC concentration shows a linear relationship in the range of 0.3 - 30 μmol / L, and the linear equation is: R / B = 0.0255[CTC] + 1.4807 (R 2 = 0.9847). This method can achieve visual quantitative detection of chlortetracycline content in meat foods, solve problems such as high cost, low sensitivity, poor selectivity and repeatability, and complex operation of existing detection methods, and has good practical application value.

[0033] (3) The present invention also studied the interference of 16 kinds of ions, 4 kinds of antibiotics, and 16 kinds of amino acids that may exist in water bodies. The results show that the response signals of the interfering substances are comparable to those of the blank group, indicating that these substances that may exist in water bodies will not affect the quantitative detection method of chlortetracycline in the meat of the present invention. The constructed ratio fluorescence probe detection system has good anti-interference ability and can accurately determine the concentration of chlortetracycline in meat. Description of the Drawings

[0034] Figure 1 It is a schematic diagram of the construction of the AuNCs fluorescence probe and the detection of chlortetracycline in Example 1.

[0035] Figure 2 (a) is the high-resolution TEM image of the Au NCs gold nanoclusters obtained in Example 1; (b) is the particle size distribution histogram of the Au NCs gold nanoclusters; (c) is the X-ray photoelectron spectroscopy of the AuNCs gold nanoclusters; (d) is the X-ray photoelectron spectroscopy of Au.

[0036] Figure 3 (a) is the maximum excitation and optimal emission fluorescence spectra of the Au NCs gold nanoclusters obtained in Example 1; (b) is the fluorescence spectra of the AuNCs gold nanoclusters at different excitation wavelengths.

[0037] Figure 4 (a) is the infrared absorption spectrum of the Au NCs gold nanoclusters obtained in Example 1; (b) is the zeta potential diagram of BSA and the AuNCs gold nanoclusters; (c) is the relationship between the fluorescence intensity of the Au NCs gold nanoclusters and the storage time.

[0038] Figure 5(a) is the fluorescence spectrum of the Au NCs gold nanocluster solution before and after reacting with the CTC solution under 350 nm fluorescence excitation; (b) is the fluorescence image of the Au NCs gold nanocluster solution before and after reacting with the CTC solution under 365 nm ultraviolet light.

[0039] Figure 6 (a) is the line graph of the fluorescence change of the system under different buffer solutions and different pH values in Example 2; (b) is the line graph of the fluorescence change of the system at different reaction times; (c) is the line graph of the fluorescence change of the system at different reaction temperatures.

[0040] Figure 7 (a) is the fluorescence spectrum of the ratio fluorescence probe with chlortetracycline concentration in the range of 0.3 - 30 μmol / L in Example 3; (b) is the linear relationship between the concentration and the fluorescence ratio with chlortetracycline concentration in the range of 0.3 - 30 μmol / L; (c) The upper and lower rows are respectively the photos of the ratio fluorescence probe system under daylight (upper) and ultraviolet light (lower) with chlortetracycline concentration in the range of 0.3 - 30 μmol / L.

[0041] Figure 8 (a) is the schematic flow chart of detecting CTC using a smart device in Example 4; (b) is the CIE chromaticity coordinates of the Au NCs gold nanocluster solution after mixing and reacting with different concentrations of CTC solution; (c) The linear relationship between R / B and CTC concentration with chlortetracycline concentration in the range of 0.3 - 30 μmol / L.

[0042] Figure 9 (a)(b) are the fluorescence spectra of different solutions under 350 nm excitation in Example 5; (c) is the change in fluorescence lifetime of Au NCs gold nanoclusters and their mixture with CTC; (d) is (I 620 / I 425 ) 0 / (I 620 / I 425 ) as a function of CTC concentration. (e) is the excitation spectrum of AuNCs gold nanoclusters and the ultraviolet absorption spectrum of CTC.

[0043] Figure 10 (a) is the selectivity bar chart of the ratio fluorescence probe for chlortetracycline detection in Example 6; (b) is the selectivity spectrum of the ratio fluorescence probe for chlortetracycline detection; (c) is the selectivity bar chart of the ratio fluorescence probe after adding different interfering substances. Detailed implementation manners

[0044] The following further analyzes and explains the implementation manners of the present invention in conjunction with the accompanying drawings. Reagents and operations not mentioned in the examples are implemented according to the conventional operations in the art.

[0045] Reagents

[0046] Tetracycline hydrochloride (TC), oxytetracycline hydrochloride (OTC), chlortetracycline hydrochloride (CTC), doxycycline hydrochloride (DOX), and chloramphenicol (CPL) were all purchased from Aladdin Reagent Co., Ltd. The purity of the tetracycline hydrochloride used was USP, and the purity of the others was 95%; MD55-5M dialysis bag (cut-off molecular weight of 1000 Da); the anions were common soluble salts, and the cations were all nitrates, purchased from Aladdin Reagent Co., Ltd.; the amino acids were purchased from Aladdin Reagent Co., Ltd.; other reagents were all of analytical grade and were used without further purification. The solutions were all prepared with secondary deionized water.

[0047] Instrument

[0048] F-7000 fluorescence spectrophotometer (Japan), TU-1901 double-beam ultraviolet-visible spectrophotometer (China), ZS90 Zeta potential analyzer (UK), SHA-C water bath thermostatic oscillator (China), FC-10A freeze dryer (China), DZF-6050 vacuum drying oven (China), FE-28 pH meter (China), H1750R high-speed desktop refrigerated centrifuge (China).

[0049] Example 1 Construction of the Au NCs gold nanocluster ratio fluorescence probe

[0050] Under stirring conditions, 0.25 g of BSA was mixed with 5 mL of AuHCl 4 (10 mM) aqueous solution and stirred vigorously for 2 minutes, then 0.5 mL of NaOH (1 M) was added, and it was placed in a 37 °C thermostatic oscillator and slowly shaken for 12 h to obtain a pale yellow transparent solution. It was purified with a 1000 Da dialysis membrane for 12 h to obtain an Au NCs gold nanocluster solution emitting red fluorescence. The gold nanocluster solution was freeze-dried to obtain the AuNCs gold nanocluster finished product, which was stored for later use at 4 °C.

[0051] Take the AuNCs gold nanocluster finished product and prepare it into an aqueous solution of gold nanoclusters (10 μg / mL). Add 100 μL of Tris-HCl (0.04 M, pH 10) buffer solution to obtain the Au NCs ratio fluorescence probe. This fluorescence probe can be used for the ratio fluorescence detection or visual detection of the chlortetracycline content in meat products. The schematic diagram of the construction and detection application of the ratio fluorescence probe is shown in Figure 1 .

[0052] Characterize the AuNCs gold nanoclusters obtained in Example 1, and the results are shown in Figure 2 . From Figure 2 a, 2b, it can be seen that the AuNCs gold nanoclusters are in the form of uniform monodisperse spheres with a particle size of 3.40 nm. The high-resolution TEM image (HRTEM) clearly resolves the lattice spacing of 0.13 nm.Figure 2 c is the X-ray photoelectron spectroscopy of the Au NCs gold nanocluster product. From Figure 2 c, it can be seen that C, O, N, Na, and Au are located at 286 eV, 532 eV, 400 eV, 1072 eV, and 64 eV respectively. The elemental peaks of C, N, and O are derived from the BSA protein respectively, the Na elemental peak is from NaOH in the synthesis conditions, and the Au peak is from the AuNCs nanoclusters. Figure 2 d is the X-ray photoelectron spectroscopy of Au 4f. The oxidation state of gold atoms was determined by X-ray photoelectron spectroscopy (XPS). Initially, the oxidation state of the gold (Au) atoms in 4 HAuC1 is Au 3+ , when BSA protein is added to the HAuC1 4 solution and stirred vigorously, most of the gold (Au) atoms are reduced from Au 3+ to Au + . Then, 1 M NaOH solution is added to the reaction mixture, and the reaction is carried out for an appropriate time under stirring and heating conditions to form BSA-Au nanocrystals. During this process, the color of the system changes from dark yellow to reddish-brown, confirming the reduction of gold atoms from Au + to Au 0 to form clusters. And some outer gold atoms have the Au + oxidation state because during the formation of Au NCs, some carbonyl functional groups containing amino acids in the BSA native protein coordinate with the outer gold atoms.

[0053] As Figure 3 shown in a, the Au NCs gold nanocluster has a maximum excitation emission spectrum at 350 nm and two emission peaks at 425 nm and 620 nm. Figure 3 b is the fluorescence emission map of the AuNCs gold nanocluster at an excitation wavelength of 310 nm - 410 nm. As Figure 3 shown in b, at an excitation wavelength of 350 nm, the Au NCs gold nanocluster has a strong fluorescence emission peak with a good peak shape and is not affected by the second harmonic peak. Therefore, 350 nm is selected as the optimal excitation wavelength.

[0054] As Figure 4 shown in the infrared absorption spectrum of the aAu NCs gold nanocluster, the peak at 1654 cm -1 corresponds to the C=O stretching vibration of the amide bond in the peptide bond, and the peak at 3448 cm -1 is due to the stretching vibration of -OH and -NH of the intermolecular hydrogen bond in the peptide. To further understand the chemical properties and stability of AuNCs, Zeta potential tests were carried out on BSA and the AuNCs gold nanocluster (see Figure 4 b). As Figure 4As shown in Fig. b, the Zeta potential value of the Au NCs gold nanoclusters synthesized based on bovine serum albumin is -42 mV, and the absolute value of its potential is greater than 30 mV, indicating that the Au NC gold nanoclusters are in a relatively stable state at this time. In addition, the change of the fluorescence intensity of the Au NCs gold nanoclusters with storage time was investigated, and the results are as Figure 4 shown in Fig. c. The synthesized Au NCs gold nanoclusters can remain stable for at least 8 weeks. The above results all confirm the successful synthesis of the AuNCs gold nanoclusters.

[0055] The fluorescence spectra and color changes under ultraviolet light of the Au NCs ratiometric fluorescence probe before and after reacting with CTC are shown in Figure 5 . As Figure 5 shown in Fig. a, under the excitation light of 350 nm, after the Au NCs ratiometric fluorescence probe reacts with CTC, the fluorescence emission band of the system at 425 nm is significantly enhanced, and the fluorescence emission band at 625 nm has no obvious change. As Figure 5 shown in Fig. b, after adding 10 μM of CTC solution, the color of the AuNCs ratiometric fluorescence probe under 365 nm ultraviolet light changes from red to purple.

[0056] Example 2 Optimization of Detection Conditions

[0057] In order to make the sensing detection performance reach the best state, in this example, the pH value of the buffer solution, the reaction time, and the reaction temperature were optimized.

[0058] Effect of buffer pH on the fluorescence change of the detection system. Add 100 μL of different types and different pH buffer solutions to 100 μL of the Au NCs gold nanocluster solution (50 μg / mL). After mixing evenly, add 10 μL of the chlortetracycline standard solution (1 mM, prepared with the corresponding buffer solution), and add ultrapure water to make up to 1 mL. After reacting at room temperature for 5 min, under the excitation of 350 nm wavelength, record the fluorescence intensity in the range of 370 - 670 nm. The selected buffer solutions are PBS, Tris-HCl, and BR, and the pH values of each buffer solution are set to 7, 8, 9, 10, 11, and 12 respectively. The fluorescence intensity ratio (F 425 / F 620 ) of each reaction system is shown in Figure 6 Fig. a. As Figure 6 shown in Fig. a, by comparing the three buffer solutions, it is found that when the Tris-HCl buffer solution is used in the system, the change of Δ(I 425 / I 620 ) is the most obvious, and it reaches the maximum at pH 10. Therefore, the Tris-HCl buffer solution with pH 10 is selected as the best detection condition.

[0059] Effect of reaction time on fluorescence change of the detection system. Add 100 μL of Tris-HCl (0.04 M, pH 10) buffer solution to 100 μL of Au NCs gold nanocluster solution (50 μg / mL). After mixing evenly, add 10 μL of chlortetracycline standard solution (1 mM, prepared with this buffer solution), and make up the volume to 1 mL with ultrapure water. Measure the fluorescence intensities at 425 nm and 620 nm when the reaction proceeds for 1, 2, 5, 8, 10, 20, 30, 40, and 50 min. As Figure 6 shown in b, the fluorescence intensity ratio at 425 nm and 620 nm is basically stable after 5 min. Therefore, the optimal reaction time is 5 min.

[0060] Effect of reaction temperature on fluorescence change of the detection system. Add 100 μL of Tris-HCl (0.04 M, pH 10) buffer solution to 100 μL of Au NCs gold nanocluster solution (50 μg / mL). After mixing evenly, add 10 μL of chlortetracycline standard solution (1 mM, prepared with this buffer solution), and make up the volume to 1 mL with ultrapure water. React at 10 °C, 20 °C, 30 °C, 40 °C, and 50 °C for 5 min respectively, and measure the fluorescence intensities at 425 nm and 620 nm. As Figure 6 shown in c, the change of the fluorescence intensity ratio at 425 nm and 620 nm is the most obvious when reacting at 30 °C. Therefore, the optimal reaction temperature is 30 °C.

[0061] It can be seen from Example 2 that the Tris-HCl buffer solution with pH 10, reaction time of 5 min, and reaction temperature of 30 °C is the optimal reaction system.

[0062] Example 3 Detection of chlortetracycline by ratio fluorescence probe

[0063] Add 100 μL of Tris-HCl (0.04 M, pH 10) buffer solution to 100 μL of Au NCs gold nanocluster solution (50 μg / mL). After mixing evenly, then add 10 μL of chlortetracycline standard solutions with different concentrations (0.3, 0.5, 1, 2, 5, 8, 10, 12, 15, 20, 25, 30 μM), make up the volume to 1 mL with ultrapure water. After incubating at 30 °C for 5 min, measure at an excitation wavelength of 350 nm, record the fluorescence emission spectrum in the range of 370 - 670 nm, obtain the fluorescence intensity values at 425 nm and 620 nm, and calculate F 425 / F 620 .

[0064] As Figure 7 shown in a, the CTC concentration is in the range of 0.3 - 30 μmol / L -1In the range of 0.3 - 30 μmol / L, as the CTC concentration increases, the fluorescence intensity of the system at 425 nm gradually increases, and when the CTC concentration reaches 30 μM, the fluorescence intensity gradually stabilizes. Figure 7 b is the linear relationship between CTC at different concentrations and the ratio of its fluorescence intensity (F 425 / F 620 ).

[0065] The results show that in the range of 0.3 - 30 μmol / L, the CTC concentration has a linear relationship with the fluorescence ratio. The linear relationship equation is F 425 / F 620 = 0.1718[CTC] + 1.2874 (R 2 = 0.9987), and the lowest detection limit is 0.18 μmol / L.

[0066] As Figure 7 shown in c, the CTC concentrations in the samples are different, and the solutions also show different colors under ultraviolet light. As the CTC concentration increases, the solution color gradually changes from red to purple, and the change is very obvious. Therefore, visual quantitative detection of chlortetracycline can be achieved through color comparison.

[0067] Example 4 Visual Quantitative Detection of Chlortetracycline

[0068] Cut the filter paper into a circle, immerse it in the prepared AuNCs gold nanocluster solution (50 μg / mL) for 0.5 hours, then take out the filter paper and dry it in a vacuum drying oven at 37 °C for 1 h to obtain a fluorescent paper chip. The schematic diagram of the visual quantitative detection of chlortetracycline is as Figure 8 shown in a.

[0069] Immerse the fluorescent paper chip in standard solutions of CTC at different concentrations (0.3, 0.5, 1, 2, 5, 8, 10, 12, 15, 20, 25, 30 μmol / L, prepared with 0.04 M, pH 10 Tris-HCl buffer solution), incubate at room temperature for 1 minute, and take pictures with a smartphone camera under sunlight and 365 nm ultraviolet light after drying. Use smartphone software (Color Collect) to collect the image of the fluorescent paper chip and convert it into the corresponding RGB values, and calculate the R / B value (where R and B are the values of the red and blue visible light images respectively). As Figure 8 shown in c, in the range of 0.3 - 30 μmol / L, the R / B value has a linear relationship with the CTC concentration. The linear equation is: R / B = 0.0255[CTC] + 1.4807 (R 2 = 0.9847), where [CTC] represents the concentration of chlortetracycline. Figure 8 b is the CIE chromaticity coordinates of the AuNCs gold nanocluster fluorescence probe solution in the presence of CTC at different concentrations.

[0070] When detecting a sample, after dropping the sample solution to be detected onto the fluorescent paper chip, the concentration of chlortetracycline in the sample to be detected can be calculated according to the R / B value.

[0071] Principle of the ratio fluorescence probe for detecting chlortetracycline in Example 5

[0072] In this example, the fluorescence quenching mechanism of gold nanoclusters was explored. As Figure 9 shown in a, after adding CTC, the fluorescence emission band of AuNCs gold nanoclusters at 620 nm hardly changed, while the fluorescence emission band at 425 nm was significantly enhanced. There are several hydrophobic binding cavities in the three-dimensional structure of BSA, and CTC can enter the hydrophobic binding cavity of BSA and then restrict its rotation, which enables BSA to sensitize CTC. Therefore, the fluorescence enhancement of the system at 425 nm is attributed to the sensitization effect of BSA on CTC. As Figure 9 shown in b, in the presence of BSA, the fluorescence emission peak intensity of CTC increased significantly, showing a strong fluorescence emission band, indicating that BSA does have a sensitization effect on CTC. Thus, it can be seen that the sensitization effect of BSA on CTC is the main reason why BSA-AuNCs can be used as a ratio fluorescence probe for CTC. Further combining the fluorescence lifetime to explore the sensing mechanism of CTC, the fluorescence lifetime measurement results are as Figure 9 shown in c. After adding CTC, the fluorescence lifetime of BSA-AuNCs changed from 7.90 ns to 7.82 ns, that is, the fluorescence lifetime of the probe hardly changed before and after adding the analyte, indicating that the fluorescence change process is accompanied by static quenching. Based on the Stern-Volmer equation to evaluate the quenching mechanism, the equation is as follows:

[0073]

[0074] F 0 and F are respectively the ratio of the fluorescence intensities without adding CTC (I 425 / I 620 ) 0 and the ratio of the fluorescence intensities after adding CTC (I 425 / I 620 ). K SV is the Stern-Volmer constant, C q is the concentration of CTC, and K q is the quenching rate constant. In Figure 9 d, (I 442 / I 624 ) 0 / (I 442 / I 624 ) has a good linear correlation with the CTC concentration. By calculating, the Stern-Volmer quenching constant (K sv ) is 0.1195 μM-1 , the quenching rate constant (K q ) is 1.51×10 13 M -1 s -1 , which is much higher than the maximum scattering collision quenching constant of biomolecules (2×10 10 M -1 s -1 ), verifying that the fluorescence change of BSA-AuNCs caused by CTC has static quenching. In addition, as Figure 9 e shows, it is observed that the fluorescence emission spectrum of BSA-AuNCs overlaps with the ultraviolet absorption spectrum of CTC, which is mainly due to the inner filter effect. To sum up, the sensing mechanism of BSA-AuNCs to CTC is attributed to the sensitization effect of BSA on CTC, and is accompanied by static quenching caused by the inner filter effect.

[0075] Example 6 Selectivity and anti-interference ability of the ratio fluorescence probe

[0076] To explore the anti-interference ability of the sensor, the following selectivity experiments were carried out. Four kinds of antibiotic interference solutions, 16 kinds of inorganic ion interference solutions, and 16 kinds of small molecule amino acid interference solutions were prepared respectively.

[0077] Antibiotic interference solution (10 μM): Chloramphenicol (CPL), Oxytetracycline (OTC), Tetracycline (TC), Doxycycline (DOX);

[0078] Common inorganic ion interference solution (100 μM): CO 3 2- , SO 4 2- , PO 4 3- , F - , Cl - , S 2- , Cd 2+ , Pb 2+ , Fe 3+ , Fe 2 + , Cr 3+ , Co 2+ , Ag + , Zn 2+ , Cu 2+ and Ni 2+ ;

[0079] Small molecule amino acid interference solution (100 μM): L-tryptophan (Trp), L-leucine (Leu), L-proline (Pro), L-cysteine (Cys), L-methionine (Met), L-isoleucine (Ile), L-glutamic acid (Glu), L-lysine (Lys), L-tyrosine (Tyr), L-serine (Ser), L-arginine (Arg), L-asparagine (Asn), L-threonine (Thr), L-alanine (Ala), glycine (Gly), L-phenylalanine (Phe).

[0080] Anti-interference detection: 100 μL of AuNCs solution (50 μg / mL) was taken respectively, 100 μL of Tris-HCl (0.04 M, pH 10) buffer solution was added, and then 100 μL of interference solution was added respectively. Ultra-pure water was added to make the total volume of the solution 1 mL. After the above mixed solution was incubated at room temperature for 5 min, its fluorescence spectrum was recorded at an excitation wavelength of 350 nm. The results of the selectivity experiment are shown in Figure 10 . Figure 10 a, 10c results show that the response signals of each interfering substance are basically the same as those of the blank group. Therefore, the interfering substances that may exist in the meat sample will not interfere with the detection of chlortetracycline, indicating that the ratio fluorescence probe constructed in this experiment has good selectivity for the detection of chlortetracycline. Figure 10 a shows that the sensitization effect of BSA on CTC is not interfered by the other 4 antibiotics, and the ratio probe has selectivity for the detection of CTC; Figure 10 b shows that at 425 nm, the fluorescence intensity of CTC is the largest due to the sensitization effect of BSA, further indicating that the ratio probe has good selectivity for the detection of CTC and can be used for the targeted detection of CTC.

[0081] Example 7 Detection of chlortetracycline content in meat samples

[0082] Based on fluorescence spectroscopy analysis technology and the standard addition method, the constructed ratio fluorescence probe was used to detect chlortetracycline in actual meat samples. The samples were purchased from local market pork and fish.

[0083] Meat sample treatment: 2.0 g of skinned pork or fish was minced, then soaked in 5.0 mL of acetonitrile / deionized water (volume ratio V / V = 2:1), and then the above mixture was mixed on a vortex mixer for 2 min and sonicated for 20 min. The above sample solution was centrifuged at 5000 rpm for 10 min and filtered through a 0.22 μm filter membrane, and then diluted 20 times with secondary distilled water as the test solution for standby.

[0084] Spiked recovery test: Take 100 μL of AuNCs gold nanocluster solution (50 μg / mL), add 100 μL of Tris-HCl (0.04 M, pH = 10) buffer solution, and then add CTC solutions with different concentrations (0, 5, 15, and 25 μmol / L, and the volume depends on the spiked amount). Then, make up the volume of the above test solution to 1 mL with distilled water, and react at 30 °C for 5 min. Finally, the mixed solution is used for fluorescence detection. The spiked recovery test at each CTC concentration is repeated 5 times in parallel.

[0085] The experimental results are shown in Table 1. The recovery rate of chlortetracycline reaches 94.60 - 98.00%, indicating that this method has a good recovery rate, and the constructed ratio fluorescence probe can achieve accurate detection of chlortetracycline in actual samples.

[0086] Table 1 Detection data of chlortetracycline content in meat samples

[0087]

[0088] In this invention, the prepared Au NCs gold nanoclusters are applied to the ratio fluorescence detection of CTC, and a ratio fluorescence probe is constructed through the inner filter effect and the sensitization effect of BSA on CTC. The results show that the AuNCs gold nanocluster ratio fluorescence probe has the advantages of simple synthesis, short response time, and low detection limit, and can achieve specific detection of CTC. In addition, this application also combines the ratio fluorescence probe with a smartphone to construct a portable smartphone platform for the visual detection of CTC, providing great potential for on-site real-time visual sensing.

Claims

1. A ratiometric fluorescent probe for detecting chlortetracycline in meat food, characterized in that: The preparation method comprises the following steps: (1-1) BSA and AuHCl4 aqueous solution were mixed and stirred vigorously, and NaOH solution was added. The mixture was shaken at a constant temperature to obtain a light yellow transparent solution; (1-2) purifying the transparent solution using a dialysis membrane to obtain a fluorescent Au NCs solution; (1-3) The Au NCs gold nanocluster solution is dried to obtain the Au NCs gold nanocluster finished product, which is stored for later use; (1-4) preparing the Au NCs gold nanocluster product into an aqueous solution, adding a buffer solution, and obtaining a ratiometric fluorescent probe; The concentration of the Au NCs gold nanoclusters in step (1-4) is 10 μg / mL, and the pH of the buffer solution is 10.

2. The ratiometric fluorescent probe for detecting chlortetracycline in meat food according to claim 1, characterized in that: In step (1-1), the mass and volume ratio of BSA, AuHCl4 and NaOH is 500 mg: 10 mL: 1 mL, the vigorous stirring time is 2 minutes, and the constant temperature oscillation condition is oscillation at 37°C for 12 hours.

3. The ratiometric fluorescent probe for detecting chlortetracycline in meat food according to claim 1, characterized in that: The dialysis membrane purification condition in step (1-2) is 1000Da dialysis membrane purification for 12h.

4. The ratiometric fluorescent probe for detecting chlortetracycline in meat food according to claim 1, characterized in that: The buffer described in step (1-4) is Tris-HCl.

5. The method for detecting chlortetracycline in meat food using the ratio fluorescence probe according to claim 1, characterized in that: The following steps are involved: (5-1) adding the test solution to the ratiometric fluorescent probe solution and incubating at 10°C to 50°C for 5 to 10 minutes; (5-2) Detection was performed at an excitation wavelength of 350 nm, and the fluorescence emission spectrum in the range of 370 to 670 nm was recorded to obtain the fluorescence intensity ratio at 425 nm and 620 nm, i.e., F 425 / F 620 ; (5-3) Calculate the concentration of chloramphenicol in the test solution using the standard curve.

6. The method for detecting chlortetracycline in meat food according to claim 5, characterized in that: The incubation conditions in step (5-1) are 30° C. and 5 min.

7. The method for detecting chlortetracycline in meat food according to claim 5, characterized in that: The standard curve is: F 425 / F 620 =0.1718[CTC]+1.2874, [CTC] represents the concentration of chlortetracycline.

8. The method for detecting chlortetracycline in meat food using the ratio fluorescence probe according to claim 1, characterized in that: The following steps are involved: (8-1) Preparation of fluorescent paper chip: Soak the filter paper in the ratiometric fluorescent probe solution, take it out, dry it and set it aside. (8-2) immersing the fluorescent paper chip prepared in step (8-1) in the test solution and incubating for 1 min; (8-3) drying the fluorescent paper chip after incubation in step (8-2), collecting the image of the fluorescent paper chip using software under a 365 nm ultraviolet lamp, performing color recognition on the collected image, and converting it into corresponding RGB values, and calculating the R / B value; (8-4) Calculate the concentration of chloramphenicol in the test solution using the standard curve.

9. The method for detecting chlortetracycline in meat food according to claim 8, characterized in that: In step (8-1), the filter paper is immersed in the ratiometric fluorescent probe solution for 30 minutes.

10. The method for detecting chlortetracycline in meat food according to claim 8, characterized in that: The standard curve is: R / B=0.0255[CTC]+1.4807, [CTC] represents the concentration of chlortetracycline.

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