CN117949421A - Application of Alizarin/Al3+ Ratio Fluorescent Probe in Detection of Ciprofloxacin - Google Patents

Abstract

The invention discloses an application of an alizarin/Al ³⁺ -based ratio fluorescent probe in ciprofloxacin detection, wherein the alizarin/Al ³⁺ -based ratio fluorescent probe is formed by mixing an alizarin solution, an AlCl ₃ solution and a HEPES buffer solution, under the condition that ciprofloxacin exists, excitation light with the wavelength of 355-365 nm is used for excitation, red fluorescence exists at 647-653nm, and blue fluorescence exists at 433-439 nm; the red fluorescence is used as a reference signal, the blue fluorescence is used as a response signal, and the ratio of the two fluorescence and the concentration of ciprofloxacin have a linear relationship, so that the method can be used for quantitative detection of ciprofloxacin; in addition, the alizarin/Al ³⁺ ratio fluorescent probe changes the color of the probe solution from red to purple to blue along with the increase of the concentration of the ciprofloxacin, so that semi-quantitative visual detection of the ciprofloxacin can be realized. The alizarin/Al ³⁺ -based ratio fluorescent probe has excellent selectivity and anti-interference capability, can realize visual detection and quantitative analysis of ciprofloxacin residues in eggs, and has good sensitivity and high accuracy.

Description

Application of Alizarin/Al3+ Ratio Fluorescent Probe in Detection of Ciprofloxacin

Technical Field

The invention belongs to the technical field of food safety detection, and particularly relates to application of an alizarin/Al ³⁺ -based ratio fluorescent probe in detecting ciprofloxacin.

Background technique

The reliance on antibiotics in the intensive breeding of laying hens has greatly increased the risk of drug residues in eggs. Ciprofloxacin (CIP) belongs to the fluoroquinolone antibiotics. It has a simple structure, a broad antibacterial spectrum, strong antibacterial activity, and a low price. It is widely used in the poultry industry to treat and prevent bacterial infections and other diseases. However, due to unreasonable use (such as failure to implement the withdrawal period in accordance with relevant national regulations) and poor degradability, CIP is very easy to remain and enter the human body through the food chain. This may cause the body to develop antibiotic resistance, causing diseases such as tendon injury, hepatotoxicity, adverse reactions of the central nervous system, and gastrointestinal dysfunction. At present, many countries have banned the use of some antibiotics in poultry farming and strictly stipulated the residue limits of antibiotics. The national food safety standard GB 31650-2019 stipulates the maximum residue limit of CIP in food and clearly prohibits its use during the egg-laying period of poultry. Therefore, establishing an accurate and sensitive CIP residue detection method is of great significance to ensure the quality and safety of eggs and promote the healthy development of my country's poultry and egg industry.

The currently developed analytical methods for detecting CIP residues mainly include high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis, enzyme-linked immunosorbent assay (ELISA), electrochemical method, etc. These methods have the disadvantages of complicated sample pretreatment steps, expensive instruments, high dependence on antibodies, and high detection costs. In addition, problems such as poor reproducibility and stability also limit the widespread application of electrochemical detection methods.

In recent years, although research on CIP fluorescence detection has made some progress at home and abroad. For example, Xia Hong et al. disclosed a method for detecting CIP using CdSe quantum dots, by adding different concentrations of ciprofloxacin to the CdSe quantum dot solution, incubating with phosphate buffer at room temperature in the dark, and exciting with light of wavelength 438nm. Since CIP will enhance the fluorescence of CdSe quantum dots through fluorescence resonance energy transfer, the fluorescence intensity at 530nm gradually increases, and the fluorescence intensity within the emission range of 450-650nm is recorded; then, a standard curve is proposed with CIP concentration as the horizontal axis and _F0 /F as the vertical axis, so as to realize the detection of CIP; wherein _F0 and F are the maximum emission intensities of the system in the absence and presence of ciprofloxacin, respectively. Liu Baoxia et al. reported a fluorescence method for detecting CIP using lanthanide coordination polymer nanoparticles (Eu/GMP NPs). Due to the strong coordination interaction between CIP and Eu ³⁺ , their fluorescence was significantly enhanced. Using Eu/GMP NPs as a fluorescent probe, the fluorescence intensity of Eu/GMP NPs in aqueous solution was measured at an excitation wavelength of 276 nm in the presence of different concentrations of CIP. Then, a standard curve was made with the fluorescence intensity of Eu/GMP NPs at 615 nm and the CIP concentration as the ordinate and abscissa, respectively, to quantitatively analyze CIP. However, the above methods are all based on fluorescent probes for single signal detection, and the detection results are easily interfered by the probe concentration, excitation wavelength and environmental factors, which reduces the detection accuracy of the analyte.

Summary of the invention

The technical problem to be solved by the present invention is to provide an application of an alizarin/Al ³⁺ -based ratio fluorescent probe in the detection of ciprofloxacin in view of the deficiencies in the above-mentioned prior art. The probe has excellent selectivity and anti-interference ability, can realize the visual detection and quantitative analysis of ciprofloxacin residues in eggs, and has good sensitivity and high accuracy.

The technical solution adopted by the present invention to solve the above-mentioned problems is:

The invention discloses an alizarin/Al ³⁺ -based ratio fluorescent probe for detecting ciprofloxacin, characterized in that the alizarin/Al ³⁺ -based ratio fluorescent probe induces alizarin aggregation to produce aggregation induced effect (AIE) through coordination of Al ³⁺ with -OH groups on the surface of alizarin, thereby causing fluorescence intensity enhancement, thereby generating stronger red fluorescence at 647-653nm; and the coordination between Al ³⁺ and ciprofloxacin will cause the intrinsic fluorescence of ciprofloxacin to be significantly enhanced, thereby generating stronger blue fluorescence at 433-439nm; with red fluorescence as a reference signal and blue fluorescence as a response signal, the ratio of the two fluorescences (the ratio of red fluorescence intensity to blue fluorescence intensity, or the ratio of blue fluorescence intensity to red fluorescence intensity) has a linear relationship with the ciprofloxacin concentration in the range of 0.1μM-15μM, thereby realizing the detection of the ciprofloxacin content in the solution to be tested.

According to the above scheme, as the concentration of ciprofloxacin increases, the color of the alizarin/Al ³⁺ ratio fluorescent probe (solution) changes from red to purple and then to blue, thereby realizing semi-quantitative visual detection of the concentration of ciprofloxacin in the test solution.

The preparation method of the above alizarin/Al ³⁺ -based ratio fluorescent probe comprises the following steps:

(1) Using water as solvent, prepare an alizarin solution with a concentration of 0.4 mg/mL to 0.6 mg/mL;

(2) Using water as solvent, prepare an AlCl ₃ solution with a concentration of 3mM-5mM;

(3) fully mixing the alizarin solution and the AlCl ₃ solution in a volume ratio of (0.7-1) 1, and allowing to stand at room temperature to obtain a mixed solution;

(4) Add HEPES buffer to the mixed solution obtained in step (3), and incubate at 55-65° C. for 10-20 minutes to obtain an alizarin/Al ³⁺ -based ratio fluorescent probe.

According to the above scheme, in step (4), the volume ratio of the mixed solution to the HEPES buffer is in the range of (0.6-0.9):1.

According to the above scheme, the concentration of HEPES buffer is 0.005-0.015 M and the pH is 4.8-5.2.

According to the above scheme, in step (4), after adding HEPES buffer, a certain amount of water can be added for dilution, and then incubated at 55-65° C. for 10-20 minutes to obtain an alizarin/Al ³⁺ -based ratio fluorescent probe (solution). The volume ratio of the added amount of water to the HEPES buffer is 1:(2-4).

On the basis of the above, the present invention provides a method for highly sensitively detecting ciprofloxacin residues in eggs based on an alizarin/Al ³⁺ -based ratio fluorescent probe, comprising the following steps:

1) Using water as solvent, prepare CIP standard solutions of different concentrations, ranging from 0 to 15 μM;

2) adding the CIP standard solutions of different concentrations described in step 1) to the alizarin/Al ³⁺ ratio fluorescent probe (solution) of the present invention in parallel, incubating at 55-65° C. for 10-20 minutes, and measuring the fluorescence intensity ratio at 433-439 nm and 647-653 nm at an excitation wavelength of 355 nm-365 nm, and establishing a standard curve between the fluorescence intensity ratio and the concentration of the CIP standard solution with the fluorescence intensity ratio as the ordinate and the concentration of the CIP standard solution as the abscissa;

3) The fluorescence intensity ratio of the sample to be tested at 433-439 nm and 647-653 nm is measured under the same conditions as in step 2), and then the CIP concentration in the sample to be tested is obtained according to the standard curve obtained in step 2).

According to the above scheme, the sample to be tested is an egg extract, and the preparation method is as follows: weigh a certain amount of egg liquid sample (whole egg liquid, egg white liquid or egg yolk liquid) of fresh eggs, vortex mix evenly; then use a mixed solution of acetonitrile and water for ultrasonic treatment, and then centrifuge to collect the supernatant, which is the egg extract, as the sample to be tested for detecting ciprofloxacin residues in eggs. Of course, it can also be diluted with a buffer solution and/or water, preferably a HEPES buffer.

Compared with the prior art, the present invention has the following beneficial effects:

1. The present invention constructs a novel alizarin/Al ³⁺ ratio fluorescent probe based on aggregation-induced emission effect (AIE) and coordination to realize visual detection of ciprofloxacin residues. In the alizarin/Al ³⁺ -based ratio fluorescent probe, Al ³⁺ triggers alizarin aggregation to cause fluorescence intensity enhancement, and its red fluorescence is used as a reference signal; the coordination between Al ³⁺ and ciprofloxacin causes a significant enhancement of the intrinsic fluorescence of ciprofloxacin, and this blue fluorescence is used as a response signal; as the concentration of ciprofloxacin increases, the probe (solution) changes from red to purple and then to blue, which provides the possibility for semi-quantitative visual analysis. Moreover, the alizarin/Al ³⁺ -based ratio fluorescent probe shows a good linear response to ciprofloxacin in the linear range of 0.01 to 10.00 μM (R ² = 0.991), and the detection limit is 6.9 nM. In addition, the alizarin/Al ³⁺ -based ratio fluorescent probe also has excellent selectivity and anti-interference ability, and shows satisfactory recovery rate and stability in the spiked recovery experiment of ciprofloxacin residues in egg samples.

2. The present invention uses ratio fluorescence detection to improve sensitivity and accuracy through self-calibration, which is more accurate and stable than single signal detection and is convenient for visual monitoring.

3. The present invention is low-cost, easy to operate, accurate and reliable, and can complete the detection in only 15 minutes, and has a good application prospect.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effect of Al ³⁺ on the fluorescence emission intensity of alizarin and the effect of CIP on the fluorescence emission intensity of alizarin/Al ³⁺ -based ratio fluorescent probe, including fluorescence spectra of four solutions a, b, c, and d and photographs under ultraviolet light; wherein a is an alizarin solution with a concentration of 0.080 mg/mL; b is the alizarin/Al ³⁺ -based ratio fluorescent probe (solution) prepared in Example 1, wherein the final concentration of alizarin is approximately 0.080 mg/mL and the final concentration of Al ³⁺ is approximately 0.776 mM; c is a CIP solution with a concentration of 0.806 μM; d is a mixed solution of the alizarin/Al ³⁺ -based ratio fluorescent probe (solution) prepared in Example 1 and a CIP solution, wherein the final concentration of CIP is 0.806 μM, the final concentration of alizarin is 0.080 mg/mL, and the final concentration of Al ³⁺ is 0.776 mM.

Figure 2 shows the schematic diagram of the coordination between alizarin and Al ³⁺ .

Figure 3 shows the hydrodynamic diameter distribution of the Al ³⁺ solution before and after the addition of the alizarin solution in Example 2; in the figure, alizarin represents the alizarin solution, and the concentration is 0.080 mg/mL; alizarin + Al ³⁺ represents the alizarin/Al ³⁺ -based ratio fluorescent probe (solution) prepared in Example 1, wherein the final concentration of alizarin is 0.080 mg/mL and the final concentration of Al ³⁺ is 0.776 mM.

Figure 4 shows the binding model of Al ³⁺ and CIP.

Figure 5 shows the hydrodynamic diameter distribution diagram of the CIP solution before and after the addition of the alizarin/Al ³⁺ -based ratio fluorescent probe in Example 2; wherein, alizarin + Al ³⁺ represents the alizarin/Al ³⁺ -based ratio fluorescent probe (solution) prepared in Example 1, wherein the final concentration of alizarin is 0.080 mg/mL and the final concentration of Al ³⁺ is 0.776 mM; alizarin + Al ³⁺ +CIP represents a mixed solution of the alizarin/Al 3+-based ratio fluorescent probe (solution) prepared in Example 1 and the CIP solution, wherein the final concentration of CIP is 0.806 μM, the final concentration of alizarin is 0.080 mg/mL, and the final concentration ^{of Al 3+ is} 0.776 mM.

FIG6 shows the fluorescence emission spectrum of the alizarin/Al ³⁺ -based ratio fluorescent probe in Example 2 after adding CIP.

FIG. 7 shows the linear correlation curve between the CIP concentration and the fluorescence intensity ratio (I ₄₃₆ /I ₆₅₀ ) in Example 2. FIG.

Detailed ways

In order to better understand the present invention, the content of the present invention is further explained below in conjunction with embodiments, but the present invention is not limited to the following embodiments.

In the following examples, the reagents and instruments used are:

Aluminum chloride hexahydrate (AlCl ₃ ·6H2O), ciprofloxacin (CIP), chloramphenicol (CHL), roxithromycin (ROX), ampicillin sodium (AMP), lincomycin hydrochloride (LIN), streptomycin (STR), azithromycin (AZI), kanamycin sulfate (KAN), gentamicin sulfate (GEN), nitrofurazone (FRZ), sodium sulfate, sodium sulfite, magnesium chloride, zinc chloride, glucose, L-histidine (L-His), and acetonitrile were all provided by Shanghai MacLean Biochemical Technology Co., Ltd., China; alizarin was provided by Xilong Technology Co., Ltd. (Shantou, Guangdong); 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and dimethyl sulfoxide were purchased from Shanghai Aladdin Chemical Reagent Co., Ltd., China; the water used for solution preparation in the experiment was ultrapure water; HEPES buffer was: 0.01 M HEPES, pH = 5.0. The above chemical reagents were all analytically pure and were not further purified unless otherwise specified.

FL 6500 fluorescence spectrophotometer (PerkinElmer Instrument Co., Ltd., USA); ML-3002T/02 precision electronic balance (accuracy 0.0001g, Mettler-Toledo International Co., Ltd.); Zeta potentiometer (Zeta, ZSU3100, UK); DF-101S constant temperature water bath (Gongyi Yuhua Instrument Co., Ltd.); KQ-250DE ultrasonic multi-frequency cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); high-speed refrigerated centrifuge (JW-3021HR).

Example 1 Construction and characterization of alizarin/Al ³⁺ ratio fluorescent probe

Alizarin solution (500 μL, 0.5 mg/mL) and AlCl ₃ solution (600 μL, 4.0 mM) were fully mixed and then allowed to stand at room temperature (24°C) for 5 min to obtain a mixed solution. Subsequently, 1500 μL of HEPES buffer (0.01 M, pH = 5.0) was added to the mixed solution, incubated at 60°C for 15 min, and then 500 μL of water was added to obtain an alizarin/Al ³⁺ -based ratio fluorescent probe (solution).

As can be seen from Figure 1, the alizarin solution exhibits a weak fluorescence signal at 650nm under an excitation wavelength of 360nm. With the addition of Al ³⁺ , the fluorescence signal of the alizarin solution at 650nm is significantly enhanced, that is, the alizarin/Al ³⁺ -based ratio fluorescence probe (solution) has significant red fluorescence at 650nm, and also exhibits significant red fluorescence under ultraviolet light. This may be due to the fact that Al ³⁺ induces alizarin aggregation by coordinating with the -OH groups on the surface of alizarin to produce an aggregation-induced effect (AIE), thereby producing stronger fluorescence. The CIP solution exhibits intrinsic fluorescence at 436nm. When it is added to the alizarin/Al ³⁺ -based ratio fluorescence probe solution, Al ³⁺ significantly enhances the fluorescence signal of CIP, while the 650nm fluorescence emission peak derived from alizarin remains essentially unchanged, which provides the possibility for ratio fluorescence detection of CIP. Accordingly, the solution photo under 365nm ultraviolet light further verifies this color transition.

Figure 3 shows the hydrodynamic diameter of alizarin alone and after the introduction of Al ³⁺ . It can be seen that the introduction of Al ³⁺ induces the aggregation of the alizarin solution, causing its average diameter to change from 25.95nm to 48.99nm. Therefore, Al ³⁺ can be added to the alizarin solution to construct a fluorescent probe, and CIP can be detected by calculating the ratio of the fluorescence signals at 436nm and 650nm.

Example 2 Method for detecting ciprofloxacin using alizarin/Al ³⁺ -based ratio fluorescent probe

500 μL of CIP standard solutions of different concentrations (0, 0.1, 0.5, 1, 2.5, 5, 7, 10, 13, 15 μM) were added to the alizarin/Al ³⁺ -based ratio fluorescent probe (solution) prepared in Example 1, and incubated at 60°C for 15 minutes. The fluorescence spectrum was recorded at room temperature under an excitation wavelength of 360 nm, and the fluorescence intensity at 436 nm and 650 nm (labeled as I ₄₃₆ and I ₆₅₀ ) was measured, where the slits of the excitation wavelength and the emission wavelength were both 5 nm.

As shown in Figure 4, Al ³⁺ inhibits the excited state intramolecular proton transfer (ESIPT) of CIP molecules by complexing with carboxyl and pyridone in CIP, thereby enhancing the fluorescence signal of CIP. It can be inferred that the response mechanism of the alizarin/Al ³⁺ -based ratio fluorescent probe to CIP may be due to the fact that Al ³⁺ further forms a complex with CIP after binding to alizarin, inhibiting the ESIPT of CIP, resulting in an enhancement of the fluorescence signal. To verify this hypothesis, its hydrodynamic diameter was also measured. As shown in Figure 5, the addition of CIP increased the average diameter of the alizarin/Al ³⁺ -based ratio fluorescent probe from 48.99nm to 88.26nm, indicating that CIP can bind to the alizarin/Al ³⁺ -based ratio fluorescent probe through electrostatic interactions.

The standard curve was fitted with the fluorescence intensity ratio of the two fluorescence signals (I ₄₃₆ /I ₆₅₀ ) as the ordinate and the CIP concentration as the abscissa. The linear fitting result is shown in FIG7 . The fitting equation is y=1.760x-0.137 (R ² =0.991, S/N=3). The linear range is between 0.01 and 10.00 μM. The detection limit is 6.9 nM, indicating that the fluorescence signal of the alizarin/Al ³⁺ -based ratio fluorescent probe of the present invention has a good linear relationship with the CIP concentration and can be used to detect the concentration of ciprofloxacin in the sample to be tested.

Example 3: Method for detecting ciprofloxacin residues in eggs using alizarin/Al ³⁺ -based ratio fluorescent probe

In order to evaluate the application potential and feasibility of the alizarin/Al ³⁺ -based ratio fluorescent probe in actual detection, the residual amount of fluoroquinolones in eggs was analyzed by the spike recovery method. 1.0 g of fresh egg whole egg liquid sample was weighed and vortexed to mix evenly. Then, 2 mL of CIP solution was added (3 times in parallel, the concentrations of CIP solution were 0, 0.5, 1.5, and 2.5 μM, respectively), and then 5 mL of acetonitrile solution (acetonitrile: water, 9:1, v/v) was added. After vortexing again, ultrasonic treatment was performed for 10 minutes, centrifuged at 4°C for 10 minutes (12000 rpm), and the supernatant was collected. Repeat the above operation 3 times, and the supernatant was collected as the egg extract, which was used as the test sample for detecting ciprofloxacin residues in eggs for later use.

500 μL of the above egg extract (CIP concentrations of 0, 0.5, 1.5, and 2.5 μM, respectively) was added to the alizarin/Al ³⁺ -based ratio fluorescent probe (solution) prepared in Example 1, and incubated at 60°C for 15 min. The fluorescence intensity at 436 nm and 650 nm was measured at an excitation wavelength of 360 nm, and the concentration of ciprofloxacin in the egg extract was calculated by substituting it into the standard curve in Example 2. The experiment was set up in 3 parallels, and the average value was taken.

The inspection results are shown in Table 1. The recovery rate of CIP in actual egg samples is 82.00% to 102.00%, and the relative standard deviation ranges from 0.06% to 0.17%, showing good recovery rate and relative standard deviation, indicating that the method for detecting ciprofloxacin residues in eggs based on alizarin/Al ³⁺ -based ratio fluorescent probe provided by the present invention performs well in FQs detection, can be used for sensitive and accurate detection of CIP, and has broad application prospects.

Table 1 Determination of CIP in egg samples

The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and changes without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims (10)

1. The application of the alizarin/Al ³⁺ -based ratio fluorescent probe in ciprofloxacin detection is characterized in that the alizarin/Al ³⁺ -based ratio fluorescent probe is formed by mixing an alizarin solution, an AlCl ₃ solution and a buffer solution, and water is used as a solvent; the alizarin/Al ³⁺ -based ratio fluorescent probe is excited by excitation light with the wavelength of 355-365 nm under the condition that ciprofloxacin exists, red fluorescence exists at 647-653nm, and blue fluorescence exists at 433-439 nm; the ratio of the fluorescence intensities of the red fluorescence and the blue fluorescence has a linear relation with the concentration of ciprofloxacin, and is used for realizing quantitative detection of the ciprofloxacin.

2. The application of the alizarin/Al ³⁺ -based ratio fluorescent probe in ciprofloxacin detection is characterized in that the alizarin/Al ³⁺ -based ratio fluorescent probe is formed by mixing an alizarin solution, an AlCl ₃ solution and a buffer solution, and water is used as a solvent; the alizarin/Al ³⁺ -based ratio fluorescent probe is excited by excitation light with the wavelength of 355-365 nm under the condition that ciprofloxacin exists, red fluorescence exists at 647-653nm, and blue fluorescence exists at 433-439 nm; the alizarin/Al ³⁺ ratio fluorescent probe changes the color of a probe solution from red to blue along with the increase of the concentration of ciprofloxacin, and is used for realizing semi-quantitative visual detection of the ciprofloxacin.

3. The use of the alizarin/Al ³⁺ -based ratio fluorescent probe according to claim 1 or 2 in ciprofloxacin detection, wherein the preparation method of the alizarin/Al ³⁺ -based ratio fluorescent probe comprises the following steps:

(1) Preparing alizarin solution with concentration of 0.4mg/mL-0.6mg/mL by taking water as a solvent;

(2) Preparing AlCl ₃ solution with the concentration of 3mM-5mM by taking water as a solvent;

(3) Fully and uniformly mixing alizarin solution and AlCl ₃ solution according to volume ratio (0.7-1) 1, and standing at room temperature to obtain mixed solution;

(4) Adding buffer solution or buffer solution and water into the mixed solution obtained in the step (3), and incubating for 10-20 minutes at 55-65 ℃ to obtain the alizarin/Al ³⁺ -based ratio fluorescent probe; the alizarin/Al ³⁺ -based ratio fluorescent probe is a solution, wherein the final concentration of alizarin is in the range of 0.05-0.10mg/mL, and the final concentration of Al ³⁺ is in the range of 0.5-1.0 mM.

4. The use of alizarin/Al ³⁺ -based ratio fluorescent probe according to claim 3 for ciprofloxacin detection, wherein the buffer is HEPES buffer with a concentration of 0.005-0.015m and a ph of 4.8-5.2.

5. The use of the alizarin/Al ³⁺ -based ratio fluorescent probe according to claim 3 in ciprofloxacin detection, wherein in step (4), the volume ratio of the mixed solution to the buffer is (0.6-0.9): 1 range; the volume ratio of the mixed solution to water is (1.8-2.7): 1.

6. The use of the alizarin/Al ³⁺ -based ratio fluorescent probe according to claim 1 or 2 in ciprofloxacin detection, characterized in that the specific application method comprises the following steps:

1) Preparing ciprofloxacin standard solutions with different concentrations by taking water as a solvent, wherein the concentration range is 0-15 mu M;

2) Respectively adding the ciprofloxacin standard solutions with different concentrations in the step 1) into the alizarin/Al ³⁺ ratio fluorescent probe, incubating for 10-20 minutes at 55-65 ℃, measuring the fluorescence intensity ratio at two places of 433-439nm and 647-653nm under the excitation wavelength of 355-365 nm, taking the fluorescence intensity ratio as an ordinate, taking the concentration of the ciprofloxacin standard solution as an abscissa, and establishing a standard curve between the fluorescence intensity ratio and the concentration of the ciprofloxacin standard solution;

3) And (3) measuring the fluorescence intensity ratio of 433-439nm and 647-653nm of the sample to be measured under the same condition as the step (2), and further obtaining the ciprofloxacin concentration in the sample to be measured according to the standard curve obtained in the step (2).

7. The application of the alizarin/Al ³⁺ -based ratio fluorescent probe in ciprofloxacin detection, wherein the sample to be detected is egg extract, and the preparation method is as follows: and (3) uniformly mixing fresh egg liquid by vortex, then carrying out ultrasonic treatment by using a mixed solution of acetonitrile and water, and centrifuging to collect supernatant, namely an egg extract, wherein the supernatant is used as a sample to be detected for detecting ciprofloxacin residues in eggs.

8. The use of the alizarin/Al ³⁺ -based ratio fluorescent probe in ciprofloxacin detection according to claim 7, wherein the egg extract is diluted with HEPES buffer as a sample to be tested for detecting ciprofloxacin residues in eggs.

9. The method for detecting ciprofloxacin residues in eggs based on alizarin/Al ³⁺ -based ratio fluorescent probe is characterized by comprising the following specific steps:

S1, preparing an alizarin/Al ³⁺ -based ratio fluorescent probe: uniformly mixing alizarin aqueous solution with concentration of 0.4mg/mL-0.6mg/mL and AlCl ₃ aqueous solution with concentration of 3mM-5mM according to volume ratio (0.7-1) 1 to obtain mixed solution; mixing the obtained mixed solution with a buffer solution, or mixing the obtained mixed solution with the buffer solution and water, and then incubating for 10-20 minutes at 55-65 ℃ to obtain the alizarin/Al ³⁺ -based ratio fluorescent probe, wherein the final concentration of alizarin is in the range of 0.05-0.10mg/mL and the final concentration of Al ³⁺ is in the range of 0.5-1.0 mM;

s2, respectively adding ciprofloxacin standard solutions with different concentrations into the alizarin/Al ³⁺ ratio fluorescent probe obtained in the step S1 in parallel, incubating at 55-65 ℃ for 10-20 minutes, measuring the fluorescence intensity ratio of 433-439nm and 647-653nm at the excitation wavelength of 355-365 nm, taking the fluorescence intensity ratio as an ordinate, taking the concentration of the ciprofloxacin standard solution as an abscissa, and establishing a standard curve between the fluorescence intensity ratio and the concentration of the ciprofloxacin standard solution;

s3, taking an egg extract of fresh eggs as a sample to be detected for detecting ciprofloxacin residues in the eggs, measuring the fluorescence intensity ratio of 433-439nm and 647-653nm of the sample to be detected under the same condition as that of the step S2, and further obtaining the ciprofloxacin concentration in the sample to be detected according to a standard curve obtained in the step S2.

10. The method for detecting ciprofloxacin residues in eggs based on alizarin/Al ³⁺ -based ratio fluorescent probes according to claim 9, wherein the linear range of the standard curve is in the range of ciprofloxacin concentration of 0.1 μm to 15 μm.