A fluorescent probe for detecting gatifloxacin and its application in food

Abstract

The invention relates to the field of fluorescent nanomaterials, and in particular to a fluorescent probe for detecting gatifloxacin and an application thereof in food. The invention uses bear bile powder as a precursor to prepare a novel biomatrix-derived carbon quantum dot, and constructs a highly selective fluorescent sensing platform, which is successfully used to detect gatifloxacin in milk and honey. The prepared carbon quantum dots show obvious concentration dependence. Under sunlight, as the concentration of the carbon quantum dots increases, the color of the solution gradually changes from light yellow to orange-yellow, and under ultraviolet light, it shows a transition from blue fluorescence to green fluorescence. By establishing a linear relationship between the fluorescence signal intensity and the gatifloxacin concentration, sensitive detection of gatifloxacin is achieved, and the detection limit can be as low as 3 μg / L, which proves the excellent performance of the fluorescent probe and provides a certain reference value for the application of biomatrix-derived carbon quantum dots in the field of food safety detection.

Description

Technical Field

[0001] The invention relates to the field of fluorescent nanomaterials, and in particular to a fluorescent probe for detecting gatifloxacin and application thereof in food. Background Art

[0002] Quinolones are a relatively new class of synthetic antibiotics with broad-spectrum bactericidal activity against Gram-positive and Gram-negative bacteria. They are widely used to treat bacterial infections in animals. However, the residues of these drugs in animals can lead to a variety of adverse reactions, including the development of antimicrobial resistance and low efficiency of antibiotic treatment for bacterial infections and gastrointestinal irritation in humans. In addition, antibiotic residues in animal-derived foods (such as meat, chicken, milk, eggs, and honey) pose serious health hazards to humans, including nephrotoxicity, ototoxicity, and damage to the digestive and nervous systems, which has become a matter of great concern and in-depth research in recent years. As one of the important fluoroquinolones (FAs), gatifloxacin (GAT) is in urgent need of developing sufficiently sensitive analytical techniques to monitor and determine its residues in food.

[0003] Although most traditional detection methods, such as high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA) and capillary electrophoresis (CE) are very sensitive, they are time-consuming and laborious, or require special and expensive instruments, and are difficult to be used for on-site detection of antibiotics. Fluorescence method has attracted wide attention due to its high sensitivity, simple equipment, and convenient operation. For example, patent technology document CN117126139A discloses a colorimetric copper ion fluorescent probe and its synthesis and application, in which the copper ion recognition group described in the method is one of norfloxacin, levofloxacin, moxifloxacin and gatifloxacin commercial drugs, and the copper ion fluorescent probe described in the invention can be used for copper ion qualitative and colorimetric detection, particularly for solution / cell / tissue / living imaging, biomarkers or sensing fields.

[0004] However, the current preparation methods of fluorescent carbon quantum dot fluorescent probes include hydrothermal method, microwave method, laser burning method, etc. These methods require a lot of energy to synthesize carbon quantum dots, and have the problems of high energy consumption, high cost and environmental pollution.

[0005] Therefore, according to the above-mentioned related technologies, it is urgent to develop a fluorescent probe for detecting gatifloxacin and its application in food. Summary of the invention

[0006] In view of this, the purpose of the present invention is to propose a fluorescent probe for detecting gatifloxacin and its application in food, so as to provide a new fluorescent probe based on biological matrix derivative bear bile powder carbon quantum dots (CDs), which has a simple preparation process, does not use toxic and complex chemical reagents, is environmentally friendly and pollution-free, has low cost and high fluorescence intensity and quantum yield, and can be used to quickly detect gatifloxacin.

[0007] Based on the above purpose, the present invention provides a fluorescent probe for detecting gatifloxacin and its application in food.

[0008] A fluorescent probe for detecting gatifloxacin, wherein the preparation method of the fluorescent probe is as follows:

[0009] Step S1. The method for preparing the fluorescent probe comprises the following steps:

[0010] Step S1. Weigh bear bile powder and dissolve it in deionized water, sonicate and vortex to fully dissolve it, to obtain a mixed solution A;

[0011] Step S2. placing the mixed solution A in a high pressure reactor, heating for reaction, and obtaining a bear bile carbon quantum dot solution;

[0012] Step S3. Cooling the bear bile carbon quantum dot solution to room temperature, transferring it to a centrifuge tube and centrifuging it to obtain a supernatant of the bear bile carbon quantum dot solution;

[0013] Step S4. Filter the supernatant of the bear bile carbon quantum dot solution, transfer the obtained filtrate into a dialysis bag, dialyze in deionized water, and freeze-dry under vacuum after dialysis to obtain a bear bile carbon quantum dot solid powder;

[0014] Step S5. Dissolve the bear bile carbon quantum dot solid powder in deionized water to obtain a fluorescent probe.

[0015] Preferably, the usage ratio of the bear bile powder and deionized water in step S1 is 0.23-0.28 g: 25-30 mL.

[0016] Preferably, in step S1, the ultrasonic time is 5-8 min, and the vortex time is 1-3 min.

[0017] Preferably, the temperature of the heating reaction in step S2 is 160-180° C., and the reaction time is 5-8 h. At this reaction temperature and reaction time, the prepared fluorescent probe has a higher fluorescence intensity and fluorescence quantum yield.

[0018] Preferably, the centrifugal speed in step S3 is 4000-4200 r / min, and the centrifugal time is 5-10 min.

[0019] Preferably, the dialysis in step S4 uses a dialysis bag with a molecular weight of 500Da, and the dialysis time is 20-24h.

[0020] Preferably, the high-pressure reactor described in step S2 is a polytetrafluoroethylene liner high-pressure reactor, and the filter membrane specification used in the filtration described in step S4 is 0.22 μm.

[0021] Preferably, the freeze-drying temperature in step S4 is -80-70°C, and the freeze-drying time is 40-48 hours.

[0022] Preferably, the concentration of the fluorescent probe in step S5 is 1-5 mg / mL. Too low a concentration will result in excessive dosage, while too high a concentration will result in excessive consumption of the analyte, both of which will increase the error in reagent detection.

[0023] A fluorescent probe for detecting gatifloxacin, wherein the fluorescent probe is used to detect the content of gatifloxacin in milk and honey.

[0024] Preferably, the process of detecting the content of gatifloxacin in milk is as follows:

[0025] Step A1. Mix 300 μL of a fluorescent probe solution with a concentration of 1-5 mg / mL and 1000 μL of a gatifloxacin solution (0-100 μM) in a 5 mL centrifuge tube, add deionized water to adjust the volume of the solution to 3 mL, vortex at room temperature for 30 seconds to allow it to react fully, and then record its fluorescence intensity;

[0026] Step A2. Treat the milk with acetonitrile, then centrifuge at 4200 rpm for 10 min, filter with a 0.22 μm microporous filter membrane, mix the treated sample with the fluorescent probe solution, react at room temperature for 30 s, test its fluorescence intensity, and substitute the value into the obtained linear regression equation to calculate the concentration of gatifloxacin in the milk.

[0027] Preferably, the process of detecting the content of gatifloxacin in honey bees is as follows:

[0028] Step A1. Mix 300 μL of a fluorescent probe solution with a concentration of 1-5 mg / mL and 1000 μL of a gatifloxacin solution (0-100 μM) in a 5 mL centrifuge tube, add deionized water to adjust the volume of the solution to 3 mL, vortex at room temperature for 30 seconds to allow it to react fully, and then record its fluorescence intensity;

[0029] Step A2. Treat the honey with acetonitrile, then centrifuge at 4200 rpm for 10 min, filter with a 0.22 μm microporous filter membrane, mix the treated sample with the fluorescent probe solution, react at room temperature for 30 s, test its fluorescence intensity, and substitute the value into the obtained linear regression equation to calculate the concentration of gatifloxacin in milk.

[0030] Beneficial effects of the present invention:

[0031] 1. The present application adopts green and environmentally friendly bear bile powder as raw material to prepare a fluorescent probe that can quickly detect gatifloxacin. The preparation process is simple, no toxic or complex chemical reagents are used, it is environmentally friendly and pollution-free, low-cost and has high fluorescence intensity and quantum yield.

[0032] 2. The fluorescent probe provided in this application, the synthetic bear bile carbon quantum dots have rich oxygen-containing functional groups and nitrogen-containing functional groups such as -OH and -NH on the surface, so they show good dispersibility in aqueous solution, and there is no obvious aggregation between particles. They can be used as an efficient fluorescent sensor in actual detection.

[0033] 3. The fluorescent probe provided in the present application shows good selectivity and sensitivity for the detection of gatifloxacin, is hardly interfered by other ions, and is linearly related to the gatifloxacin content within a certain range. Therefore, it can be used to detect the content in actual samples of milk and honey, and has a very broad application prospect.

[0034] 4. The fluorescent probe provided in the present application remains basically stable in a high-concentration ion environment, and has a good fluorescence effect in a wide pH range (3-13). It also has strong photobleaching properties, and the fluorescence intensity will not change significantly even when the light source is continuously irradiated for 90 minutes.

[0035] 5. The fluorescent probe provided in this application has a short detection time and is simple to operate. The reaction can be completed quickly within 30 seconds and can be operated at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 The figure is a schematic diagram of the preparation and detection principle of the fluorescent probe for detecting gatifloxacin involved in the present invention;

[0038] Figure 2Characterization diagrams of the bear bile powder carbon quantum dots CDs involved in Example 1 of the present invention are transmission electron microscopy, X-ray diffraction and infrared spectrum;

[0039] Figure 3 This is an X-ray photoelectron spectrum of the bear bile powder carbon quantum dots CDs involved in Example 1 of the present invention;

[0040] Figure 4 This is a diagram showing the optimization of synthesis conditions of bear bile powder carbon quantum dots CDs involved in Example 1 of the present invention;

[0041] Figure 5 This is a fluorescence spectrum diagram of the bear bile powder carbon quantum dots CDs involved in Example 1 of the present invention for investigating stability;

[0042] Figure 6 The fluorescence spectra of the bear bile powder carbon quantum dots CDs involved in Example 1 of the present invention at different excitation wavelengths (400-520nm) (a), and the ultraviolet absorption spectra and optimal fluorescence excitation and emission spectra of the bear bile powder carbon quantum dots CDs, gatifloxacin, and CDs / gatifloxacin involved in the present invention (b);

[0043] Figure 7 This is a comparison diagram of the solution colors of the bear bile powder carbon quantum dots CDs involved in Example 1 of the present invention under sunlight and ultraviolet light;

[0044] Figure 8 This is a fluorescence spectrum diagram of the bear bile powder carbon quantum dots CDs at different concentrations involved in Example 1 of the present invention;

[0045] Fig. 9 The fluorescence intensity of the bear bile powder carbon quantum dots CDs involved in Example 1 of the present invention and the fluorescence reflection spectrum of gatifloxacin at different concentrations and a linear relationship diagram are shown;

[0046] Fig.10 This is a comparison chart of the interference and selectivity effects of different antibiotics, metal ions and additives on the fluorescence intensity of bear bile powder carbon quantum dots CDs solution involved in Example 1 of the present invention. DETAILED DESCRIPTION

[0047] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0048] Example 1: A method for preparing a fluorescent probe for detecting gatifloxacin, comprising the following steps:

[0049] Step S1. Weigh 0.25 g of bear bile powder and dissolve it in 25 mL of deionized water, sonicate for 5 min, and vortex for 1 min to fully dissolve it to obtain a mixed solution A;

[0050] Step S2. Place the mixed solution A in a polytetrafluoroethylene-lined autoclave, heat and react at 180° C. for 5 h to obtain a bear bile carbon quantum dot solution;

[0051] Step S3. The bear bile carbon quantum dot solution was cooled to room temperature, transferred to a 50 mL centrifuge tube, and centrifuged at 4200 r / m for 5 min to remove some large solid impurities to obtain the bear bile carbon quantum dot solution supernatant;

[0052] Step S4. Filter the supernatant of the bear bile carbon quantum dot solution with a 0.22 μm filter membrane to filter out small particulate impurities, and transfer the obtained filtrate into a dialysis bag with a molecular weight of 500 Da, dialyze in deionized water for 24 hours to remove small molecules, and freeze-dry at -80°C under vacuum for 48 hours to obtain a bear bile carbon quantum dot solid powder;

[0053] Step S5. Dissolve the bear bile carbon quantum dot solid powder in deionized water to obtain a fluorescent probe with a concentration of 1 mg / mL.

[0054] like Figure 2 As shown in (a), the bear bile powder carbon quantum dots CDs prepared in Example 1 are spherical, have good dispersibility and high crystallinity. Figure 2 (a) The inset in the upper left corner shows that the synthesized bear bile powder carbon quantum dots CDs have a clear lattice structure with a lattice spacing of 0.19 μm, which is similar to the interplanar spacing of graphene;

[0055] like Figure 2 As shown in (b), the particle size of the bear bile powder carbon quantum dots CDs prepared in Example 1 is mainly distributed between 3.81-5.85 nm, and the average particle size is 4.60 nm;

[0056] like Figure 2 (c) shows the XRD analysis spectrum of the bear bile powder carbon quantum dots CDs prepared in Example 1. It can be seen that a broad diffraction peak appears at the position of 2θ=22.02°, corresponding to the 101 and 110 crystal planes of graphene carbon, indicating that the bear bile powder carbon quantum dots CDs prepared in Example 1 contain a graphite-like structure;

[0057] like Figure 2 (d) shows the infrared spectrum of the bear bile powder carbon quantum dots CDs prepared in Example 1, 3345 cm -1 The infrared absorption peak at 2929 cm-1 may be attributed to the stretching vibration of OH or NH single bonds. -1 2863.86cm -1 The infrared absorption peak at 1706cm is caused by the stretching vibration of CH. -1 The infrared absorption peak at 1648cm is caused by the stretching vibration of C=O. -1The infrared absorption peak at 1540cm comes from the stretching vibration of C=C. -1 The peak at 1448 cm -1 and 1373cm -1 The peak at 1195 cm-1 may be related to the CH bending vibration. -1 The peak at 1044 cm-1 may be related to the stretching vibration of CO. -1 The peak at 608 cm may be related to CO stretching vibration or CN stretching vibration. -1 and 525cm -1 The peak at may be related to the CH bending vibration of the aromatic ring. The above results show that the surface of bear bile powder carbon quantum dots CDs is rich in various functional groups, which makes bear bile powder carbon quantum dots CDs have good water solubility.

[0058] like Figure 3 As shown in (a), the three characteristic peaks centered at 284.04, 399.21, and 531.35 eV can be attributed to the binding energies of C1s, N1s, and O1s, respectively, indicating that the CDs prepared in Example 1 contain C, N, and O elements;

[0059] Figure 3 (b) is a high-resolution spectrum of C1s, showing three fitting peaks at 284.05, 285.6 and 287.75 eV, belonging to CC / C=C, CO and C=O, respectively, indicating the formation of unsaturated C=C double bonds in the bear bile powder carbon quantum dots CDs prepared in Example 1;

[0060] and Figure 3 The N1s spectrum of (c) has two absorption peaks at 399.07 and 401.07 eV, representing -NH and CNC / C-NH bonds, respectively;

[0061] Figure 3 (d) is the high-resolution spectrum of O1s. The two fitting peaks at 531 and 531.9 eV represent three oxygen-containing functional groups, C-OH, C=O and -OH, which confirm the existence of carbonyl. The above results are also in good agreement with the results of FT-IR infrared spectroscopy.

[0062] like Figure 4 As shown, Figure 4 (a) It can be seen that within the synthesis time range of 3-10h, when the synthesis time is 5h, the prepared bear bile powder carbon quantum dots CDs have the strongest fluorescence intensity, and the fluorescence intensity shows a significant downward trend under the conditions of shorter and longer reaction times;

[0063] Figure 4(b) shows the effect of different solvent volumes on the synthesis with the same material mass. It is found that the prepared bear bile powder carbon quantum dots CDs have the strongest fluorescence intensity at a ratio of 0.25g-30mL, and the fluorescence intensity shows a significant downward trend under conditions of less and more feed ratios. Therefore, we choose 0.25g bear bile powder and 30mL water to react at 180℃ for 5h as the preparation method of bear bile powder carbon quantum dots CDs.

[0064] like Figure 5 As shown in (a), the anti-photobleaching fluorescence intensity is almost unchanged when irradiated with ultraviolet light for 90 minutes at the optimal excitation wavelength; the fluorescence spectra of the bear bile powder carbon quantum dots CDs involved in Example 1 of the present application in salt solutions of different concentrations Figure 5 (b) shows that the fluorescent probe prepared in this application is 0-2 mol·L -1 It can remain stable in saline solution;

[0065] like Figure 5 As shown in (c), bear bile powder carbon quantum dots CDs have good fluorescence intensity under different pH values ​​(3-13);

[0066] In addition, the effect of excitation wavelength on fluorescence intensity was also investigated: e.g. Figure 6 As shown in (a), as the excitation wavelength changes from 400nm to 520nm, the emission peak gradually moves to a longer wavelength, red-shifts, and the intensity peak decreases. Therefore, the prepared bear bile powder carbon quantum dot CDs have the characteristics of excitation wavelength dependence;

[0067] like Figure 6 As shown in (b), there is an absorption peak at 200nm in the UV spectrum of bear bile powder carbon quantum dots CDs, which is attributed to the n-π * jump;

[0068] The bear bile powder carbon quantum dot CDs involved in Example 1 of the present application not only show excitation wavelength dependence, but also concentration dependence: In order to study the concentration dependence of bear bile powder carbon quantum dot CDs, a series of bear bile powder carbon quantum dot CDs solutions with concentrations (0.25, 0.5, 1, 2, 4, 6, 9, 12, 15 mg / mL) were prepared, such as Figure 7 As shown in (a), it can be clearly observed that the color of the bear bile powder carbon quantum dots CDs solution gradually deepens (from light yellow to orange-yellow) under sunlight; and Figure 7 As shown in (b), under ultraviolet light, it can be clearly observed that the color of the bear bile powder carbon quantum dots CDs solution gradually deepens (from blue fluorescence to green fluorescence).

[0069] like Figure 8As shown, the optimal excitation wavelength and the maximum emission wavelength were measured using a fluorescence spectrophotometer, and it was found that with the increase in concentration, the optimal excitation wavelength changed from 359 nm to 457 nm, and the corresponding maximum emission wavelength changed from 444 nm to 545 nm.

[0070] Example 2: A method for preparing a fluorescent probe for detecting gatifloxacin, comprising the following steps:

[0071] Step S1. Weigh 0.23 g of bear bile powder and dissolve it in 25 mL of deionized water, sonicate for 5 min, and vortex for 1 min to fully dissolve it to obtain a mixed solution A;

[0072] Step S2. Place the mixed solution A in a polytetrafluoroethylene-lined autoclave, heat and react at 160° C. for 5 h to obtain a bear bile carbon quantum dot solution;

[0073] Step S3. The bear bile carbon quantum dot solution was cooled to room temperature, transferred to a 50 mL centrifuge tube, and centrifuged at 4000 r / m for 5 min to remove some large solid impurities to obtain the bear bile carbon quantum dot solution supernatant;

[0074] Step S4. Filter the supernatant of the bear bile carbon quantum dot solution with a 0.22 μm filter membrane to filter out small particulate impurities, and transfer the obtained filtrate into a dialysis bag with a molecular weight of 500 Da, dialyze in deionized water for 20 hours to remove small molecules, and freeze-dry at -80°C under vacuum for 40 hours to obtain a bear bile carbon quantum dot solid powder;

[0075] Step S5. Dissolve the bear bile carbon quantum dot solid powder in deionized water to obtain a fluorescent probe with a concentration of 1 mg / mL.

[0076] Example 3: A method for preparing a fluorescent probe for detecting gatifloxacin, comprising the following steps:

[0077] Step S1. Weigh 0.25 g of bear bile powder and dissolve it in 26 mL of deionized water, sonicate for 6 min, and vortex for 2 min to fully dissolve it to obtain a mixed solution A;

[0078] Step S2. Place the mixed solution A in a polytetrafluoroethylene-lined autoclave, heat and react at 165° C. for 6 h to obtain a bear bile carbon quantum dot solution;

[0079] Step S3. The bear bile carbon quantum dot solution was cooled to room temperature, transferred to a 50 mL centrifuge tube, and centrifuged at 4050 r / m for 6 min to remove some large solid impurities to obtain the bear bile carbon quantum dot solution supernatant;

[0080] Step S4. Filter the supernatant of the bear bile carbon quantum dot solution with a 0.22 μm filter membrane to filter out small particulate impurities, and transfer the obtained filtrate into a dialysis bag with a molecular weight of 500 Da, dialyze in deionized water for 22 hours to remove small molecules, and freeze-dry at -78°C under vacuum for 42 hours to obtain a bear bile carbon quantum dot solid powder;

[0081] Step S5. Dissolve the bear bile carbon quantum dot solid powder in deionized water to obtain a fluorescent probe with a concentration of 2 mg / mL.

[0082] Example 4: A method for preparing a fluorescent probe for detecting gatifloxacin, comprising the following steps:

[0083] Step S1. Weigh 0.26 g of bear bile powder and dissolve it in 28 mL of deionized water, sonicate for 7 min, and vortex for 2 min to fully dissolve it to obtain a mixed solution A;

[0084] Step S2. Place the mixed solution A in a polytetrafluoroethylene-lined autoclave, heat and react at 170° C. for 7 h to obtain a bear bile carbon quantum dot solution;

[0085] Step S3. The bear bile carbon quantum dot solution was cooled to room temperature, transferred to a 50 mL centrifuge tube, and centrifuged at 4100 r / m for 8 min to remove some large solid impurities to obtain the bear bile carbon quantum dot solution supernatant;

[0086] Step S4. Filter the supernatant of the bear bile carbon quantum dot solution with a 0.22 μm filter membrane to filter out small particulate impurities, and transfer the obtained filtrate into a dialysis bag with a molecular weight of 500 Da, dialyze in deionized water for 23 hours to remove small molecules, and freeze-dry at -74°C under vacuum for 44 hours to obtain a bear bile carbon quantum dot solid powder;

[0087] Step S5. Dissolve the bear bile carbon quantum dot solid powder in deionized water to obtain a fluorescent probe with a concentration of 3 mg / mL.

[0088] Example 5: A method for preparing a fluorescent probe for detecting gatifloxacin, comprising the following steps:

[0089] Step S1. Weigh 0.28 g of bear bile powder and dissolve it in 30 mL of deionized water, sonicate for 8 min, and vortex for 3 min to fully dissolve it to obtain a mixed solution A;

[0090] Step S2. Place the mixed solution A in a polytetrafluoroethylene-lined autoclave, heat and react at 180° C. for 8 h to obtain a bear bile carbon quantum dot solution;

[0091] Step S3. The bear bile carbon quantum dot solution was cooled to room temperature, transferred to a 50 mL centrifuge tube, and centrifuged at 4200 r / m for 10 min to remove some large solid impurities to obtain the bear bile carbon quantum dot solution supernatant;

[0092] Step S4. Filter the supernatant of the bear bile carbon quantum dot solution with a 0.22 μm filter membrane to filter out small particulate impurities, and transfer the obtained filtrate into a dialysis bag with a molecular weight of 500 Da, dialyze in deionized water for 24 hours to remove small molecules, and freeze-dry at -70°C under vacuum for 48 hours to obtain a bear bile carbon quantum dot solid powder;

[0093] Step S5. Dissolve the bear bile carbon quantum dot solid powder in deionized water to obtain a fluorescent probe with a concentration of 5 mg / mL.

[0094] Example 6: A fluorescent probe for detecting gatifloxacin is used to determine gatifloxacin in milk, comprising the following steps:

[0095] Take 300 μL of the bear bile powder carbon quantum dot CDs solution in Example 1, add 1000 μL of gatifloxacin solution of different concentrations (0-100 μM) thereto, make the solution volume to 3 mL with deionized water, vortex for 30 s at room temperature to allow it to react fully, and measure and record the fluorescence intensity of the mixed solution.

[0096] like Fig. 9 As shown in (a), as the concentration of gatifloxacin increases from 0.01 to 100 μM, the fluorescence intensity increases rapidly, indicating that the bear bile powder carbon quantum dots CDs respond well to gatifloxacin. In order to investigate the relationship between gatifloxacin concentration and fluorescence intensity, a graph is plotted with F / F0 as the ordinate, as shown in Fig. 9 As shown in (b), in the range of 0.01-250 μM, F / F0 has been showing an upward trend. Fig. 9 As shown in (c), when the concentration of gatifloxacin was in the range of 0.01-100 μM, F / F0 showed a good linear relationship with the concentration of gatifloxacin, and the regression equation was y=0.0534x+1.0342, with a correlation coefficient R 2 =0.992, according to the formula LOD=3σ / k (where σ is the standard deviation of the fluorescence intensity value, k is the slope of the standard curve), the detection limit of the sensor is calculated to be 3μg / L, which can detect gatifloxacin sensitively and specifically;

[0097] The milk was pretreated to remove excess protein and fat. The sample was treated with acetonitrile, then centrifuged at 4200rpm for 10min and filtered with a 0.22μm microporous filter membrane. The actual sample solution was mixed with bear bile powder carbon quantum dots CDs and reacted at room temperature for 30s. The fluorescence intensity was tested and the value was substituted into the linear regression equation obtained above to calculate the concentration of gatifloxacin in the milk.

[0098] Example 7: A fluorescent probe for detecting gatifloxacin The determination of gatifloxacin in honey comprises the following steps:

[0099] Take 300 μL of the bear bile powder carbon quantum dot CDs solution in Example 1, add 1000 μL of gatifloxacin solution of different concentrations (0-100 μM) thereto, make the volume of the solution to 3 mL with deionized water, vortex for 30 s at room temperature to allow it to react fully, and measure and record the fluorescence intensity of the mixed solution;

[0100] The honey was pretreated to remove excess protein and fat. The sample was treated with acetonitrile, then centrifuged at 4200rpm for 10min and filtered with a 0.22μm microporous filter membrane. The actual sample solution was mixed with bear bile powder carbon quantum dots CDs and reacted at room temperature for 30s. The fluorescence intensity was tested and the value was substituted into the linear regression equation obtained above to calculate the concentration of gatifloxacin in honey.

[0101] like Fig.10 As shown in (a), common antibiotics (fluoroquinolones, chloramphenicol, macrolides, sulfonamides, and cephalosporins), common metal ions (K + , Fe 2+ 、Na + , Cu 2+ , Ca 2+ 、Zn 2+ and Mg 2+ The effects of various antibiotics, metal ions and additives (glucose, sucrose and ascorbic acid, etc.) and common additives (glucose, sucrose and ascorbic acid, etc.) on the fluorescence intensity of carbon dots were investigated. First, various antibiotics, metal ions and additives with a concentration of 1 mM and 50 μM gatifloxacin were added to the carbon dot solution, and it was found that at the optimal excitation wavelength of 365 nm, the fluorescence intensity of the detection system was significantly enhanced only when gatifloxacin was present; then, Fig.10 (b) It can be seen that even in a system with a concentration 10 times higher than that of gatifloxacin, the fluorescence intensity did not change significantly, indicating that the detection system can still accurately detect gatifloxacin even in a complex detection environment. This shows that the detection system has high selectivity and strong anti-interference ability for GAT.

[0102] Performance Test:

[0103] The recovery test of the spiked samples was carried out to evaluate the accuracy and precision of the fluorescent probe prepared in Example 1 of the present invention for detecting the content of ciprofloxacin in milk and honey. The results are shown in Table 3:

[0104] Table 1 Summary of relevant instruments and sources involved in the experiment

[0105]

[0106]

[0107] Table 2 Summary of relevant reagents and sources involved in the experiment

[0108] Instrument Name Instrument source Chloramphenicol (CHL, concentration: 99%) Shanghai Yuanye Biotechnology Co., Ltd. Roxithromycin (ROX, concentration: 99%) Shanghai Yuanye Biotechnology Co., Ltd. Thiamphenicol (THI, purity: 99%) Shanghai Yuanye Biotechnology Co., Ltd. Cephalothin (CEP, purity: 99%) Shanghai Yuanye Biotechnology Co., Ltd. Trimethoprim (TMP, purity: 99%) Shanghai Yuanye Biotechnology Co., Ltd. Sulfamethoxazole (SMX, purity: 99%) Shanghai Yuanye Biotechnology Co., Ltd. Sulfapyridine (SPD, purity: 99%) Shanghai Yuanye Biotechnology Co., Ltd. Cefpodoxime Proxetil (CPX, purity: 99%) Shanghai Yuanye Biotechnology Co., Ltd. Purified water Hangzhou Wahaha Group Co., Ltd. Bear Bile Powder Zixi County Kangxiong Industrial Co., Ltd. Gatifloxacin (GAT, purity: 98%) Shanghai MacLean Biochemical Technology Co., Ltd. Glucose (Glu, purity: 99%) Shanghai MacLean Biochemical Technology Co., Ltd. Sucrose (Suc, purity: 99%) Shanghai MacLean Biochemical Technology Co., Ltd. Ascorbic acid (AA, purity: 99%) Shanghai MacLean Biochemical Technology Co., Ltd. <![CDATA[Ferrous chloride (FeCl2, analytical pure)]]> Tianjin Hengxing Chemical Reagent Co., Ltd. <![CDATA[Copper chloride (CuCl2, analytical pure)]]> Tianjin Hengxing Chemical Reagent Co., Ltd. Potassium bromide (KBr, analytical grade) Tianjin Hengxing Chemical Reagent Co., Ltd. <![CDATA[Calcium chloride (CaCl2, analytical pure)]]> Tianjin Hengxing Chemical Reagent Co., Ltd. <![CDATA[Zinc chloride (ZnCl2, analytical reagent grade)]]> Tianjin Hengxing Chemical Reagent Co., Ltd. <![CDATA[Magnesium chloride (MgCl2, analytical reagent grade)]]> Tianjin Hengxing Chemical Reagent Co., Ltd. Sodium chloride (NaCl, analytical grade) Tianjin Hengxing Chemical Reagent Co., Ltd.

[0109] Table 3 Test results (n=3)

[0110]

[0111]

[0112] Data Analysis:

[0113] As can be seen from Table 3, the recovery rate of gatifloxacin is between 98% and 104%, and the RSD is no more than 4.5%, indicating that the fluorescent probe provided by the present invention is accurate and reliable in detecting the content of ciprofloxacin in food, and has potential application value in the simple and rapid detection of ciprofloxacin in complex samples.

[0114] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0115] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A fluorescent probe for detecting gatifloxacin, characterized in that: The preparation method of the fluorescent probe comprises the following steps: Step S1. Weigh bear bile powder and dissolve it in deionized water, sonicate and vortex to fully dissolve it, to obtain a mixed solution A; Step S2. placing the mixed solution A in a high pressure reactor, heating for reaction, and obtaining a bear bile carbon quantum dot solution; Step S3. Cooling the bear bile carbon quantum dot solution to room temperature, transferring it to a centrifuge tube and centrifuging it to obtain a supernatant of the bear bile carbon quantum dot solution; Step S4. Filter the supernatant of the bear bile carbon quantum dot solution, transfer the obtained filtrate into a dialysis bag, dialyze in deionized water, and freeze-dry under vacuum after dialysis to obtain a bear bile carbon quantum dot solid powder; Step S5. Dissolve the bear bile carbon quantum dot solid powder in deionized water to obtain a fluorescent probe.

2. The fluorescent probe for detecting gatifloxacin according to claim 1, characterized in that: The usage ratio of the bear bile powder and deionized water in step S1 is 0.23-0.28 g: 25-30 mL, the ultrasonic time in step S1 is 5-8 min, and the vortex time is 1-3 min.

3. The fluorescent probe for detecting gatifloxacin according to claim 1, characterized in that: The high-pressure reactor described in step S2 is a polytetrafluoroethylene liner high-pressure reactor, and the temperature during the heating reaction in step S2 is 160-180° C., and the reaction time is 5-8 hours.

4. The fluorescent probe for detecting gatifloxacin according to claim 1, characterized in that: The rotation speed during the centrifugation in step S3 is 4000-4200 r / min, and the centrifugation time is 5-10 min.

5. The fluorescent probe for detecting gatifloxacin according to claim 1, characterized in that: The dialysis in step S4 uses a dialysis bag with a molecular weight of 500Da, the dialysis time is 20-24h, and the filter membrane specification used in the filtration in step S4 is 0.22μm.

6. The fluorescent probe for detecting gatifloxacin according to claim 1, characterized in that: The freeze drying temperature in step S4 is -80-70°C, and the freeze drying time is 40-48h.

7. The fluorescent probe for detecting gatifloxacin according to claim 1, characterized in that: The concentration of the fluorescent probe in step S5 is 1-5 mg / mL.

8. The fluorescent probe for detecting gatifloxacin according to any one of claims 1 to 7, characterized in that: The fluorescent probe is used to detect the content of gatifloxacin in milk and honey.

9. The fluorescent probe for detecting gatifloxacin according to claim 8, characterized in that: The process of detecting the content of gatifloxacin in the milk is as follows: Step A1. Mix 300 μL of a fluorescent probe solution with a concentration of 1-5 mg / mL and 1000 μL of a gatifloxacin solution (0-100 μM) in a 5 mL centrifuge tube, add deionized water to adjust the volume of the solution to 3 mL, vortex at room temperature for 30 seconds to allow it to react fully, and then record its fluorescence intensity; Step A2. Treat the milk with acetonitrile, then centrifuge at 4200 rpm for 10 min, filter with a 0.22 μm microporous filter membrane, mix the treated sample with the fluorescent probe solution, react at room temperature for 30 s, test its fluorescence intensity, and substitute the value into the obtained linear regression equation to calculate the concentration of gatifloxacin in the milk.

10. The fluorescent probe for detecting gatifloxacin according to claim 8, characterized in that: The process of detecting the content of gatifloxacin in the honey bee is as follows: Step A1. Mix 300 μL of a fluorescent probe solution with a concentration of 1-5 mg / mL and 1000 μL of a gatifloxacin solution (0-100 μM) in a 5 mL centrifuge tube, add deionized water to adjust the volume of the solution to 3 mL, vortex at room temperature for 30 seconds to allow it to react fully, and then record its fluorescence intensity; Step A2. Treat the honey with acetonitrile, then centrifuge at 4200 rpm for 10 min, filter with a 0.22 μm microporous filter membrane, mix the treated sample with the fluorescent probe solution, react at room temperature for 30 s, test its fluorescence intensity, and substitute the value into the obtained linear regression equation to calculate the concentration of gatifloxacin in milk.

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