Ratio fluorescence probe for detecting enrofloxacin, preparation of ratiometric fluorescence probe and ratiometric fluorescence detection analysis method of enrofloxacin

Abstract

The invention discloses a ratiometric fluorescent probe for detecting enrofloxacin. The ratiometric fluorescent probe is obtained by packaging gold nanoclusters (AuNCs) in an MOFs (Metal-Organic Frameworks) material based on a metal organic framework, the gold nanocluster AuNCs takes glutathione as a precursor and is prepared from gold chloride trihydrate; the MOFs material is formed by assembling a metal ion Zn < 2 + > and an organic ligand 2-methylimidazole. The obtained ratiometric fluorescence probe is suitable for ratiometric fluorescence detection of an enrofloxacin-containing sample, especially an animal-derived food sample, the detection method is convenient and rapid, the detection result is accurate and stable, and qualitative and quantitative analysis is facilitated. An enrofloxacin ratiometric fluorescence detection analysis method constructed by using the ratiometric fluorescence probe is short in detection time, low in detection cost and beneficial to practical application and popularization.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence detection, in particular to a ratio fluorescence probe for detecting enrofloxacin and a preparation thereof, as well as an enrofloxacin ratio fluorescence detection and analysis method constructed by using the probe. Background Art

[0002] Enrofloxacin (ENRO) is a chemically synthesized third-generation quinolone drug, which is widely used in animal husbandry due to its broad-spectrum antibacterial properties. However, with the overuse and abuse of ENRO, bacteria have developed antibiotic resistance, so different countries have established maximum residue limits (MRLs) for antibiotics in animal-derived foods, requiring the MRL range of enrofloxacin to be between 100μg / kg and 300μg / kg. In order to effectively control the residues of ENRO in food, the detection of ENRO content is crucial.

[0003] At present, the detection methods for ENRO include high performance liquid chromatography, gas chromatography-mass spectrometry, enzyme-linked immunosorbent assay, electrochemical method, etc. Although these methods have the advantages of high detection sensitivity and accuracy, they have the disadvantages that the instruments used are expensive, professional personnel are required to guide the operation, the test samples need to be pretreated, and the operation steps are complicated. Therefore, the development of a highly sensitive, fast and simple ENRO detection method will have important application value.

[0004] Fluorescent probes based on fluorescent nanoparticles have attracted extensive attention due to their sensitivity, simplicity, low background and stability. Gold nanoclusters (AuNCs), as the most widely used metal nanoparticles, have quantum confinement and surface enhancement effects. In addition, their luminescence properties can be adjusted by size, structure and surface modification, making them a new type of luminescent material in the detection field. However, the low quantum yield, susceptibility to interference and instability of AuNCs limit the practical application of AuNCs. As the aggregation-induced emission (AIE) characteristics of nanoclusters have been confirmed, it has been found that when AuNCs are confined or encapsulated in a porous structure, the spatial confinement causes the fluorescent particles to be in an aggregated state, resulting in a great constraint on the intramolecular rotation. Therefore, the excited state molecules can only decay back to the ground state through radiation, thereby significantly enhancing the fluorescence and reducing the fluorescence quenching effect. However, only using the signal change of a single fluorescence channel of the fluorescent probe for detection will bring errors and instability to the results, and in the fluorescence analysis method for detecting enrofloxacin, a fluorescent probe of gold nanoclusters AuNCs with high sensitivity and selectivity has not yet been developed. Summary of the invention

[0005] In order to solve the deficiencies of the above-mentioned prior art, the present invention provides a ratio fluorescence probe for the qualitative and quantitative detection and analysis of enrofloxacin, which has the advantages of high sensitivity and high selectivity, and the detection results are accurate and stable; the ratio fluorescence detection and analysis method constructed using the probe can realize simple and rapid screening of samples to be tested, with short detection time and low detection cost, which is conducive to practical application and promotion.

[0006] To achieve the above-mentioned purpose, the ratio fluorescence probe for detecting enrofloxacin provided by the present invention is obtained by encapsulating gold nanoclusters AuNCs in a metal organic framework-based MOFs material; the gold nanoclusters AuNCs are prepared from gold chloride trihydrate using glutathione as a precursor; the MOFs material utilizes metal ions Zn 2+ and the organic ligand 2-methylimidazole.

[0007] The present invention encapsulates gold nanoclusters AuNCs prepared from specific precursors in metal organic frameworks MOFs based on the AIE effect. The obtained probe significantly improves the fluorescence performance of AuNCs and has strong emission fluorescence at two emission wavelengths. The two emission wavelengths can provide a self-correction function, so that it can be used as a ratio fluorescence probe for detecting enrofloxacin. By simultaneously recording different fluorescence emission signals of two channels, more reliable sensing results can be achieved, thereby showing excellent sensitivity and selectivity, and overcoming the errors and instability brought to the detection results by the signal changes of a single fluorescence channel of the fluorescent probe.

[0008] As a limitation of the above technical solution, the ratiometric fluorescent probe has maximum emission wavelengths at 420 nm and 600 nm respectively when excited by a fluorescence excitation wavelength of 280 nm.

[0009] As a limitation of the above technical solution, the gold nanoclusters AuNCs are prepared by mixing gold chloride trihydrate and glutathione in a molar ratio of 1:(1.5-2) in water, heating the mixture to 70-80° C. and stirring for reaction, and are denoted as GSH-AuNCs.

[0010] As a limitation of the above technical solution, gold chloride trihydrate is first added to water, and then glutathione solution is added after sufficient mixing, and the mixture is heated to 70-80° C. and stirred for reaction for 24-25 hours to obtain a GSH-AuNCs solution.

[0011] AuNCs with glutathione as precursor ensured the significant enhancement of the emission peak of the ratiometric fluorescence probe at 420nm, which was beneficial to improve the sensitivity and selectivity of the ratiometric fluorescence probe.

[0012] As a limitation of the above technical solution, the ratio fluorescent probe is prepared by reacting zinc acetate dihydrate, 2-methylimidazole, and GSH-AuNCs in a mass ratio of (123-136):(272-300):1.

[0013] From metal ion Zn 2+ The MOFs material with fixed crystal structure assembled with organic ligand 2-methylimidazole (2-MI) has the advantages of large surface area, uniform and adjustable pore size and high adsorption performance, which have significant effects on encapsulating AuNCs and enhancing their fluorescence yield. In addition, zinc acetate with low toxicity and low corrosion is selected as the raw material, and no specific temperature is required, and it can be carried out at room temperature.

[0014] The present invention provides a method for preparing the ratiometric fluorescent probe for detecting enrofloxacin as described above, comprising the following preparation steps:

[0015] Step a, 5 mL of 20 mM gold chloride trihydrate solution, 1.5 mL of 100 mM glutathione solution and 40-45 mL of water were mixed, the mixture was heated to 70-80° C. and stirred for 24-25 h to obtain a GSH-AuNCs solution, which was stored at 4° C. for subsequent use;

[0016] Step b, 0.15 g zinc acetate dihydrate and 0.33 g 2-methylimidazole were respectively dissolved in 7.5 mL methanol, the two solutions were mixed, and then a GSH-AuNCs solution containing 1.1 mg GSH-AuNCs was added, and the reaction was carried out by magnetic stirring for 1 to 1.5 h. The product was centrifuged, washed, and dried at 60 to 70 ° C to obtain powdered AuNCs@ZIF-8, which was then redissolved in methanol to obtain a ratiometric fluorescent probe for detecting enrofloxacin.

[0017] Improve the preparation of ratiometric fluorescent probes, optimize the fluorescence properties of ratiometric fluorescent probes, ensure the accuracy, sensitivity and stability of detection, and make the preparation operation simpler and safer, which is conducive to promotion and application.

[0018] At the same time, the present invention also provides a ratio fluorescence detection and analysis method for enrofloxacin, which uses the ratio fluorescence probe as described above in the ratio fluorescence detection and analysis process of enrofloxacin.

[0019] As a limitation of the above technical solution, the sample containing enrofloxacin to be detected is prepared into a solution, the ratio fluorescence probe is added, mixed evenly and incubated at room temperature in the dark for 1 min, and then the fluorescence intensity at 420 nm and 600 nm is measured under the excitation wavelength of 280 nm, respectively, and recorded as F 420 、F 600 , according to F 420 / F 600The quantitative relationship between the ratio and the enrofloxacin concentration was used to analyze the enrofloxacin content in the sample.

[0020] As a limitation of the above technical solution, the ratiometric fluorescent probe is a methanol solution of AuNCs@ZIF-8 with a concentration of 1 to 3 mg / mL.

[0021] As a limitation of the above technical solution, the mass ratio of the solution to be detected to the added amount of the ratio fluorescent probe is 1 to 2:1, and the concentration of enrofloxacin in the solution to be detected is 0 to 100 μg / L.

[0022] The ratio fluorescence probe of the present invention is suitable for ratio fluorescence detection of samples containing enrofloxacin, especially animal-derived food samples. The detection method is convenient and fast, the detection result is accurate and stable, and it is convenient for qualitative and quantitative analysis.

[0023] In summary, the ratiometric fluorescent probe and ratiometric fluorescent detection and analysis method of the present invention have the following advantages:

[0024] 1. Using glutathione as a precursor to synthesize gold nanoclusters AuNCs, encapsulated in metal organic framework-based MOFs materials, a ratiometric fluorescent probe was obtained, which not only significantly improved the fluorescence performance of AuNCs and increased the fluorescence intensity by 6 times, but also had strong fluorescence intensities at two emission wavelengths of 420nm and 600nm. When the ratiometric fluorescent probe reacted with enrofloxacin, the fluorescence intensity at the 420nm emission wavelength changed significantly, which can improve the accuracy and sensitivity of enrofloxacin detection.

[0025] 2. The constructed enrofloxacin ratio fluorescence detection and analysis method achieves more reliable sensing results by simultaneously recording the different fluorescence emission signals of two channels, thus showing excellent sensitivity and stability; compared with the immune method, there is no need to prepare antibodies, saving time and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 , the colors of AuNCs and AuNCs@ZIF-8 of Example 1 under sunlight;

[0027] Figure 2 , images of AuNCs and AuNCs@ZIF-8 of Example 1 under 280nm fluorescence;

[0028] Figure 3 , the full wavelength of fluorescence emission of AuNCs of Example 1 at different excitation wavelengths;

[0029] Figure 4 , the full wavelength of fluorescence emission of AuNCs and AuNCs@ZIF-8 of Example 1 at 280 nm;

[0030] Figure 5 , fluorescence spectra of AuNCs@ZIF-8 in Example 1 reacting with different concentrations of ENRO;

[0031] Figure 6 , the linear relationship between AuNCs@ZIF-8 of Example 1 and different concentrations of ENRO

[0032] Figure 7 , Fluorescence stability of AuNCs and AuNCs@ZIF-8 in Example 1 within 40 days

[0033] Figure 8 , the AuNCs sample photo of Example 2;

[0034] Fig. 9 , the full wavelength of fluorescence emission of AuNCs@ZIF-8 in Example 2 at different excitation wavelengths;

[0035] Fig.10 , the full wavelength of fluorescence emission of AuNCs and AuNCs@ZIF-8 at 260 nm and the fluorescence graph of the samples of Example 2, wherein a represents AuNCs and b represents AuNCs@ZIF-8;

[0036] Fig.11 , the ratio fluorescence detection and analysis method constructed by the present invention selectively analyzes 8 single antibiotics, namely ENRO, PEF, CIP, NOR, MBF, OFX, EX, and GAT, and each antibiotic + ENRO (the detection concentration of the 8 antibiotics involved is 100 μg / L);

[0037] Fig.12 , the effect of ENRO alone and a mixed system containing ENRO and 7 antibiotics (FQs) on the fluorescence intensity of the AuNCs@ZIF-8 ratiometric fluorescent probe of the present invention (the detection concentration of the 8 antibiotics involved was 100 μg / L);

[0038] Fig.13 , Fluorescence spectra of AuNCs@ZIF-8 reacting with different concentrations of ENRO in the comparative example. DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The raw materials used in the following examples and comparative examples are typical products purchased from the market.

[0041] Example 1

[0042] This embodiment relates to the preparation of a ratiometric fluorescent probe for detecting enrofloxacin, and an enrofloxacin ratiometric fluorescent detection and analysis method constructed using the probe.

[0043] The ratiometric fluorescent probe was prepared as follows:

[0044] Step a, first add gold chloride trihydrate HAuCl4·3H2O (5 mL, 20 mM) into 44.5 mL of ultrapure water, mix thoroughly and then add glutathione GSH solution (1.5 mL, 100 mM), heat the mixture to 70 °C and stir for 24 h, collect the obtained GSH-AuNCs solution and store it at 4 °C for subsequent use.

[0045] Step b, accurately weigh 0.15g zinc acetate dihydrate and 0.33g 2-methylimidazole (2-MI), respectively, add them into two centrifuge tubes each containing 7.5mL methanol, ultrasonicate for 10min (40KHz) until completely dissolved, then mix the two solutions and add 5mL GSH-AuNCs solution (containing 1.1mg GSH-AuNCs), mix well and react with magnetic stirring for 1h, the obtained product is centrifuged at 10000rpm, washed with methanol 3 times to remove the unreacted solution, and then dried at 60°C to obtain powdered AuNCs@ZIF-8, which is then redissolved in methanol to obtain a ratiometric fluorescent probe for detecting enrofloxacin.

[0046] like Figure 1 , Figure 2 As shown, the synthesized gold nanoclusters AuNCs, namely GSH-AuNCs solution, is light yellow and its fluorescent color is bright yellow; the solution prepared by AuNCs@ZIF-8 is also light yellow and its fluorescent color is brownish yellow.

[0047] like Figure 3 , Figure 4 As shown in Figure 2, by measuring the fluorescence full wavelength of GSH-AuNCs at different excitation wavelengths, it was found that the maximum excitation wavelength of GSH-AuNCs was 280 nm. After fixing the excitation wavelength at 280 nm, the maximum emission wavelengths were measured to be 420 nm and 620 nm, and F 620 / F 420 The ratio is between 2 and 10. The maximum emission wavelengths of AuNCs@ZIF-8 (2 mg / mL) obtained after encapsulation by MOFs materials are 420 nm and 600 nm, respectively, and the fluorescence intensity at 600 nm is significantly improved. Compared with GSH-AuNCs, the fluorescence intensity increases by 6 times, and F 600 / F 420The ratio also increased significantly, about 22 times. This may be because the structure of ZIF-8 restricts the intramolecular motion of AuNCs, causing AuNCs to aggregate and induce the AIE effect of AuNCs.

[0048] The ratiometric fluorescence detection analysis method of enrofloxacin using AuNCs@ZIF-8 is as follows:

[0049] 100 μL of ENRO standard solution with different concentrations (0, 0.1, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg / L) was dropped into different microwells of a 96-well microplate, and then 50 μL of AuNCs@ZIF-8 (1 mg / mL) was added to each well and mixed. After incubation at room temperature in the dark for 1 min, the fluorescence images and fluorescence spectra were collected by a microplate reader under the conditions of an excitation wavelength of 280 nm, an emission scanning range of 380-680 nm, and a slit width of 10 nm, see Figures 5 to 7 .

[0050] like Figure 5 As shown in the figure, with the increase of ENRO concentration, the fluorescence emission peak of AuNCs@ZIF-8 at 420nm gradually increased, while the fluorescence emission peak at 600nm remained unchanged. Therefore, this ratio fluorescence probe can be used for quantitative detection and analysis of ENRO.

[0051] like Figure 6 As shown in Figure 2, with the increase of ENRO concentration, the fluorescence response value F of AuNCs@ZIF-8 to ENRO 420 / F 600 The linear relationship is good between 0.1 and 100 μg / L (R 2 =0.992,p<0.05).

[0052] like Figure 7 As shown, the fluorescence intensity of AuNCs and AuNCs@ZIF-8 remained basically stable in the measurements every five days, and the fluorescence intensity after 40 days changed by 10% compared with the initial value, proving that both have good fluorescence stability. The fluorescence analysis detection method of ENRO is constructed in this way, which can enhance the reliability and stability of the method.

[0053] Although only one set of preparation condition parameters is given in the above embodiment, each preparation condition parameter can be adjusted within an appropriate range in actual preparation application. Specifically, the concentration and amount of gold chloride trihydrate solution and glutathione solution can be adjusted in step a according to the molar ratio of gold chloride trihydrate to glutathione of 1:(1.5-2), the amount of water can be appropriately increased or decreased according to the principle of facilitating the reaction, the reaction heating temperature can be 70-80°C, and the reaction time can be in the range of 24-25h; the amount of zinc acetate dihydrate and 2-methylimidazole and the amount of GSH-AuNCs solution in step b can be adjusted according to the mass ratio of zinc acetate dihydrate, 2-methylimidazole, and GSH-AuNCs of (123-136):(272-300):1, the reaction time can be 1-1.5h, and the drying temperature can be in the range of 60-70°C.

[0054] In addition, when the ratiometric fluorescent probe AuNCs@ZIF-8 is used in the ratiometric fluorescence detection analysis of enrofloxacin, the concentration used can be 1-3 mg / mL, and the mass ratio of the solution to be detected to the ratiometric fluorescent probe can be 1-2:1.

[0055] Example 2

[0056] This example relates to the effects of different preparation operations of GSH-AuNCs on the ratiometric fluorescent probe AuNCs@ZIF-8.

[0057] Example 2-1

[0058] First, GSH solution (1.5 mL, 100 mM) was added to 44.5 mL of ultrapure water. After thorough mixing, HAuCl4·3H2O solution (5 mL, 20 mM) was added. The mixture was heated to 70 °C and stirred for 24 h. The obtained GSH-AuNCs solution was collected and stored at 4 °C for subsequent use.

[0059] Example 2-2

[0060] HAuCl4·3H2O solution (5 mL, 20 mM) and GSH solution (1.5 mL, 100 mM) were simultaneously added to 44.5 mL of ultrapure water. After thorough mixing, the mixture was heated to 70 °C and stirred for 24 h. The obtained GSH-AuNCs solution was collected and stored at 4 °C for subsequent use.

[0061] like Figures 8 to 10As shown, the GSH-AuNCs solutions synthesized by the two preparation methods of Examples 2-1 and 2-2 have the same properties, both are milky white under sunlight and pink under fluorescence; the AuNCs@ZIF-8 prepared by these two GSH-AuNCs (the other preparation operations of AuNCs@ZIF-8 are the same as in Example 1) have a maximum excitation wavelength of 260nm, a maximum emission wavelength of 370nm and 610nm, and a maximum fluorescence intensity of 1917, and also have the characteristics of strong emission fluorescence at two emission wavelengths, but there is a distance from the optimal excitation wavelength of ENRO, which will affect the sensitivity of detection, and the fluorescence intensity is lower than that of Example 1.

[0062] By comparing Examples 1 and 2, it can be seen that during the synthesis process, the addition order and mixing steps of HAuCl4·3H2O and GSH are different, which will affect the fluorescence intensity and fluorescence color of AuNCs, and will also affect the optimal excitation wavelength and emission wavelength. The operation of Example 1 is the optimal solution. Compared with Example 1, the detection results of Example 2 will have certain instability.

[0063] Example 3

[0064] This example relates to the specificity of the ratiometric fluorescent probe AuNCs@ZIF-8 of the present invention for the detection of enrofloxacin.

[0065] Seven quinolone antibiotics (FQs) with similar structures to ENRO were selected: pefloxacin (PEF), ciprofloxacin (CIP), norfloxacin (NOR), marbofloxacin (MBF), ofloxacin (OFX), enoxacin (EX), and gatifloxacin (GEM).

[0066] At an excitation wavelength of 280 nm, the recognition of ENRO and seven other antibiotics by the ratiometric fluorescent probe of Example 1 was determined, and the results were compared based on the F 420 / F 600 The ratio of was used to evaluate the specific recognition ability of the AuNCs@ZIF-8 ratio fluorescence probe of Example 1 for ENRO.

[0067] The results are as follows Fig.11 As shown, the probe AuNCs@ZIF-8 has a single substance F for ENRO and seven other antibiotics. 420 / F 600The values ​​are ENRO (100%), PEF (37.60%), CIP (36.71%), NOR (27.11%), MBF (23.07%), OFX (21.03%), EX (21.05%) and GEM (21.37%), which shows that the probe AuNCs@ZIF-8 has good selectivity for ENRO. At the same time, when ENRO coexists with other antibiotics, the fluorescence response deviates slightly from the fluorescence response of ENRO alone, with an RSD of 3.81% (n=3). More importantly, when ENRO and seven FQs coexist in a system, with each antibiotic concentration of 100 μg / L, the recognition ability of the probe AuNCs@ZIF-8 for ENRO is not significantly different from the results of detecting ENRO alone, such as Fig.12 shown.

[0068] The above results show that the ratiometric fluorescent probe AuNCs@ZIF-8 of the present invention has good anti-interference ability for the detection of ENRO and can be used to detect the residual ENRO in complex environments.

[0069] Comparative Example

[0070] This comparative example involves the effect of synthesizing AuNCs using bovine serum albumin (BSA) as a precursor to prepare the ratiometric fluorescent probe AuNCs@ZIF-8 for detecting ENRO.

[0071] Synthesis method of BSA-AuNCs: BSA (5 mL, 50 mg / mL) was used as a precursor and mixed with HAuCl4·3H2O (5 mL, 10 mM) solution, stirred at 25°C for 2 min, adjusted the pH to 12 with 1 M NaOH, and incubated with magnetic stirring at room temperature for 12 h to obtain BSA-AuNCs solution. The subsequent preparation of the ratiometric fluorescent probe AuNCs@ZIF-8 was the same as in Example 1.

[0072] The recognition performance of the probe AuNCs@ZIF-8 synthesized with BSA as the precursor for ENRO is shown in the following figure. Fig.13 As shown in the figure, the ratiometric fluorescent probe AuNCs@ZIF-8 prepared by BSA-AuNCs did not react significantly with ENRO. Under the excitation wavelength of 270nm, the emission peak enhancement at 420nm was not significant, indicating that the binding reaction with ENRO was very weak, which had certain limitations for the subsequent construction of ENRO detection method. In addition, the AuNCs synthesized by this method only had one emission peak at 420nm and could not constitute a ratiometric fluorescence detection system, which limited the detection range and sensitivity of the method.

[0073] In summary, the ratiometric fluorescent probe AuNCs@ZIF-8 of the present invention significantly improves the fluorescence performance of AuNCs, and has dual emission wavelengths at 420nm and 600nm. When it reacts with enrofloxacin, the fluorescence intensity at the emission wavelength of 420nm changes significantly, while the emission wavelength at 600nm remains unchanged. On this basis, a ratiometric fluorescence detection and analysis method for detecting enrofloxacin is successfully constructed. The two emission wavelengths of the ratiometric fluorescent probe provide a self-correction function, and by simultaneously recording different fluorescence emission signals of two channels, more reliable sensing results are achieved, thereby showing excellent sensitivity, selectivity and stability.

Claims

1. A ratiometric fluorescent probe for detecting enrofloxacin, characterized in that: The ratio fluorescence probe is obtained by encapsulating gold nanoclusters AuNCs in a metal organic framework (MOFs) material; the gold nanoclusters AuNCs are prepared from gold chloride trihydrate using glutathione as a precursor; the MOFs material utilizes metal ions Zn 2+ and the organic ligand 2-methylimidazole.

2. The ratiometric fluorescent probe for detecting enrofloxacin according to claim 1, characterized in that: The ratiometric fluorescent probe has maximum emission wavelengths at 420 nm and 600 nm respectively when excited by a fluorescence excitation wavelength of 280 nm.

3. The ratiometric fluorescent probe for detecting enrofloxacin according to claim 1, characterized in that: The gold nanoclusters AuNCs are prepared by mixing gold chloride trihydrate and glutathione in a molar ratio of 1:(1.5-2) in water, heating the mixture to 70-80° C. and stirring for reaction, and are denoted as GSH-AuNCs.

4. The ratiometric fluorescent probe for detecting enrofloxacin according to claim 3, characterized in that: First, gold chloride trihydrate was added to water, and then glutathione solution was added after being fully mixed. The mixture was heated to 70-80° C. and stirred for reaction for 24-25 h to obtain a GSH-AuNCs solution.

5. The ratiometric fluorescent probe for detecting enrofloxacin according to claim 3, characterized in that: The ratiometric fluorescent probe was prepared by reacting zinc acetate dihydrate, 2-methylimidazole, and GSH-AuNCs in a mass ratio of (123-136):(272-300):

1.

6. A method for preparing a ratiometric fluorescent probe for detecting enrofloxacin according to any one of claims 1 to 5, characterized in that: The method comprises the following preparation steps: Step a, 5 mL of 20 mM gold chloride trihydrate solution, 1.5 mL of 100 mM glutathione solution and 40-45 mL of water were mixed, the mixture was heated to 70-80° C. and stirred for 24-25 h to obtain a GSH-AuNCs solution, which was stored at 4° C. for subsequent use; Step b, 0.15 g zinc acetate dihydrate and 0.33 g 2-methylimidazole were respectively dissolved in 7.5 mL methanol, the two solutions were mixed, and then a GSH-AuNCs solution containing 1.1 mg GSH-AuNCs was added, and the reaction was carried out by magnetic stirring for 1 to 1.5 h. The product was centrifuged, washed, and dried at 60 to 70 ° C to obtain powdered AuNCs@ZIF-8, which was then redissolved in methanol to obtain a ratiometric fluorescent probe for detecting enrofloxacin.

7. A ratio fluorescence detection and analysis method for enrofloxacin, characterized in that: The ratiometric fluorescent probe according to any one of claims 1 to 6 is used in the ratiometric fluorescent detection analysis of enrofloxacin.

8. The ratio fluorescence detection and analysis method of enrofloxacin according to claim 7, characterized in that: The sample containing enrofloxacin to be tested was prepared into a solution, and the ratio fluorescence probe was added. After mixing evenly, it was incubated at room temperature in the dark for 1 min, and then the fluorescence intensity at 420 nm and 600 nm was measured under the excitation wavelength of 280 nm, respectively, and recorded as F 420 、F 600 , according to F 420 / F 600 The quantitative relationship between the ratio and the enrofloxacin concentration was used to analyze the enrofloxacin content in the sample.

9. The ratio fluorescence detection and analysis method of enrofloxacin according to claim 8, characterized in that: The ratiometric fluorescent probe is a methanol solution of AuNCs@ZIF-8 with a concentration of 1 to 3 mg / mL.

10. The ratio fluorescence detection and analysis method of enrofloxacin according to claim 9, characterized in that: The mass ratio of the solution to be detected to the ratio fluorescent probe added is 1-2:1, and the concentration of enrofloxacin in the solution to be detected is 0-100 μg / L.

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