Alizarin / Al 3+ Application of ratiometric fluorescent probe in the detection of ciprofloxacin
By using an alizarin/Al3+ group ratio fluorescent probe, the aggregation-induced effect caused by the coordination of Al3+ with the -OH group on the surface of alizarin and the coordination effect of ciprofloxacin are utilized to solve the problems of complexity and accuracy in the detection of ciprofloxacin in the existing technology, and realize low-cost, high-sensitivity visualization and quantitative analysis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ANHUI AGRICULTURAL UNIVERSITY
- Filing Date
- 2024-01-16
- Publication Date
- 2026-06-02
AI Technical Summary
Existing methods for detecting ciprofloxacin suffer from cumbersome sample pretreatment steps, expensive instruments, high dependence on antibodies, high detection costs, poor reproducibility and stability, and single signal detection is easily affected by probe concentration, excitation wavelength and environmental factors, leading to a decrease in detection accuracy.
An alizarin/Al3+ group ratio fluorescent probe was used. The aggregation-induced effect (AIE) caused by the coordination of Al3+ with the -OH group on the surface of alizarin enhances fluorescence. Combined with the coordination of Al3+ with ciprofloxacin, the concentration of ciprofloxacin was detected by the ratio of red fluorescence to blue fluorescence, so as to achieve visualization and quantitative analysis.
It achieves high sensitivity, accuracy and anti-interference ability for ciprofloxacin residues, is low in cost and easy to operate, and can realize semi-quantitative visual detection and quantitative analysis of ciprofloxacin in egg samples. The detection limit is 6.9 nM, and the recovery rate and stability are good.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of food safety testing technology, specifically relating to alizarin / Al. 3+ Application of ratiometric fluorescent probes in the detection of ciprofloxacin. Background Technology
[0002] Ciprofloxacin (CIP) belongs to the fluoroquinolone class of antibiotics. It is simple in structure, has a broad antibacterial spectrum, strong antibacterial activity, and is inexpensive, making it widely used in poultry farming for the treatment and prevention of bacterial infections. However, due to improper use and poor degradation, CIP easily remains and enters the human body through the food chain. This can lead to antibiotic resistance, causing tendon damage, liver toxicity, adverse reactions in the central nervous system, and gastrointestinal disorders. Therefore, establishing accurate and sensitive methods for detecting CIP residues is of great significance for ensuring the safety and quality of eggs.
[0003] 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), and electrochemical methods. These methods either have drawbacks such as cumbersome sample pretreatment steps and expensive equipment, or high dependence on antibodies and high detection costs. Furthermore, poor reproducibility and stability also limit the widespread application of electrochemical detection methods.
[0004] In recent years, although some progress has been made in the research on CIP fluorescence detection both domestically and internationally, for example, Xia Hong et al. disclosed a method for detecting CIP using CdSe quantum dots. By adding different concentrations of ciprofloxacin to a CdSe quantum dot solution, incubating it in the dark at room temperature with phosphate buffer, and then exciting it with light at a wavelength of 438 nm, the fluorescence intensity at 530 nm gradually increased due to CIP enhancing CdSe quantum dot fluorescence through fluorescence resonance energy transfer. The fluorescence intensity in the emission range of 450-650 nm was recorded. A standard curve was then constructed with CIP concentration as the x-axis and F0 / F as the y-axis, thus realizing the detection of CIP. F0 and F represent 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). Because CIP and Eu... 3+The strong coordination interaction between them significantly enhances their fluorescence. Using Eu / GMP NPs as fluorescent probes, the fluorescence intensity of Eu / GMP NPs in aqueous solution with different concentrations of CIP was measured at an excitation wavelength of 276 nm. Then, a standard curve was plotted with the fluorescence intensity of Eu / GMP NPs at 615 nm and the CIP concentration as the ordinate and abscissa, respectively, for quantitative analysis of CIP. However, the above methods are all based on fluorescent probes with single-signal detection, and the detection results are easily affected by probe concentration, excitation wavelength, and environmental factors, which reduces the accuracy of analyte detection. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide an alizarin / Al solution that addresses the shortcomings of the prior art. 3+ The application of ratiometric fluorescent probes in the detection of ciprofloxacin demonstrates that the probes possess excellent selectivity and anti-interference capabilities, enabling the visual detection and quantitative analysis of ciprofloxacin residues in eggs with high sensitivity and accuracy.
[0006] The technical solution adopted by the present invention to solve the above-mentioned problems is as follows:
[0007] Alizarin 3+ The application of a ratio-modified fluorescent probe in the detection of ciprofloxacin, characterized in that the alizarin / Al 3+ The base ratio fluorescent probe is obtained through Al 3+ Al coordinates with the -OH groups on the alizarin surface, inducing alizarin aggregation and producing an aggregation-induced effect (AIE), which enhances fluorescence intensity, resulting in strong red fluorescence at 647-653 nm; while Al 3+ The coordination interaction with ciprofloxacin significantly enhances the intrinsic fluorescence of ciprofloxacin, producing strong blue fluorescence at 433-439 nm. Using 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 0.1-15 μM, thereby enabling the detection of ciprofloxacin content in the test solution.
[0008] According to the above scheme, the alizarin / Al 3+ As the concentration of ciprofloxacin increases, the ratiometric fluorescent probe (solution) changes from red to purple and then to blue, enabling semi-quantitative visual detection of ciprofloxacin concentration in the test solution.
[0009] The above alizarin / Al 3+ The preparation method of the base ratio fluorescent probe includes the following steps:
[0010] (1) Prepare an alizarin solution with a concentration of 0.4-0.6 mg / mL using water as a solvent;
[0011] (2) Prepare an AlCl3 solution with a concentration of 3-5 mM using water as a solvent;
[0012] (3) Mix the alizarin solution and AlCl3 solution thoroughly at a volume ratio of (0.7-1):1, and let stand at room temperature to obtain a mixed solution;
[0013] (4) Add HEPES buffer to the mixed solution obtained in step (3), and incubate at 55-65 °C for 10-20 minutes to obtain alizarin / Al 3+ Base ratio fluorescent probe.
[0014] 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.
[0015] According to the above protocol, the concentration of HEPES buffer is 0.005-0.015 M, and the pH is 4.8-5.2.
[0016] 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 ℃ for 10-20 minutes to obtain alizarin / Al. 3+ A ratio-modified fluorescent probe (solution) is prepared. The volume ratio of water to HEPES buffer is 1:(2-4).
[0017] Based on the above, the present invention provides a method based on alizarin / Al 3+ A method for highly sensitive detection of ciprofloxacin residues in eggs using a base ratio fluorescent probe includes the following steps:
[0018] 1) Using water as a solvent, prepare CIP standard solutions of different concentrations, ranging from 0 to 15 μM;
[0019] 2) The CIP standard solutions of different concentrations described in step 1) are added in parallel to the alizarin / Al solution of the present invention. 3+ After incubating the ratiometric fluorescent probe (solution) at 55-65 ℃ for 10-20 minutes, the fluorescence intensity ratio at 433-439 nm and 647-653 nm was measured at an excitation wavelength of 355-365 nm. A standard curve was established 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.
[0020] 3) Under the same conditions as in step 2), measure the fluorescence intensity ratio at 433-439 nm and 647-653 nm of the sample to be tested, and then obtain the CIP concentration in the sample to be tested based on the standard curve obtained in step 2).
[0021] According to the above scheme, the sample to be tested is egg extract, and the preparation method is as follows: Weigh a certain amount of fresh egg liquid sample (whole egg liquid, egg white liquid, or egg yolk liquid are all acceptable), vortex mix evenly; then sonicate with a mixture of acetonitrile and water, and then centrifuge to collect the supernatant, which is the egg extract, used as the sample to detect ciprofloxacin residues in eggs. Of course, it can also be diluted with buffer solution and / or water, preferably HEPES buffer.
[0022] Compared with the prior art, the beneficial effects of the present invention are:
[0023] 1. This invention constructs a novel alizarin / Al based on aggregation-induced emission (AIE) and coordination effects. 3+ A ratiometric fluorescent probe enabled the visual detection of ciprofloxacin residues. In this alizarin / Al 3+ In the base ratio fluorescent probe, Al 3+ Alizarin aggregation is triggered, leading to increased fluorescence intensity; its red fluorescence serves as a reference signal. 3+ The coordination with ciprofloxacin resulted in a significant enhancement of ciprofloxacin's intrinsic fluorescence, with this blue fluorescence serving as a response signal. As the concentration of ciprofloxacin increased, the probe (solution) changed from red to purple and then to blue, enabling semi-quantitative visual analysis. Furthermore, this alizarin / Al... 3+ The ratiometric fluorescent probe exhibited a good linear response to ciprofloxacin in the linear range of 0.01–10.00 μM (R0). 2 =0.991), with a detection limit of 6.9 nM. Furthermore, this alizarin / Al 3+ The base ratio fluorescent probe also has excellent selectivity and anti-interference ability, and showed satisfactory recovery rate and stability in the spiked recovery experiment of ciprofloxacin residue in egg samples.
[0024] 2. This 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 also convenient for visual monitoring.
[0025] 3. This invention is low in cost, easy to operate, accurate and reliable, and can complete the detection in just 15 minutes, and has a very good application prospect. Attached Figure Description
[0026] Figure 1 Al was displayed 3+ Effects of alizarin fluorescence emission intensity and CIP on alizarin / Al3+ The effect of the ratio of the fluorescent probe on the fluorescence emission intensity was investigated, including the fluorescence spectra and ultraviolet photographs of four solutions (a, b, c, and d); where a is an alizarin solution with a concentration of 0.080 mg / mL; b is the alizarin / Al solution prepared in Example 1. 3+ A ratiometric fluorescent probe (solution) with alizarin at a final concentration of approximately 0.080 mg / mL, and Al 3+ The final concentration was approximately 0.776 mM; c was a CIP solution with a concentration of 0.806 μM; d was the alizarin / Al solution prepared in Example 1. 3+ A mixed solution of a base-ratio fluorescent probe (solution) and CIP solution, wherein the final concentration of CIP is 0.806 μM, the final concentration of alizarin is 0.080 mg / mL, and Al... 3+ The final concentration was 0.776 mM.
[0027] Figure 2 It showed alizarin and Al 3+ Coordination principle diagram.
[0028] Figure 3 Example 2 shows Al 3+ Hydrodynamic diameter distribution before and after the addition of alizarin solution; in the figure, alizarin represents the alizarin solution with a concentration of 0.080 mg / mL; alizarin + Al 3+ Alizarin / Al prepared as a representative example of Example 1 3+ A ratiometric fluorescent probe (solution) with alizarin at a final concentration of 0.080 mg / mL and Al... 3+ The final concentration was 0.776 mM.
[0029] Figure 4 Al was displayed 3+ A model combining CIP.
[0030] Figure 5 The addition of alizarin / Al to the CIP solution in Example 2 is shown. 3+ Hydrodynamic diameter distribution before and after the base ratio fluorescent probe; where alizarin + Al 3+ Alizarin / Al prepared as a representative example of Example 1 3+ A ratiometric fluorescent probe (solution) with alizarin at a final concentration of 0.080 mg / mL and Al... 3+ The final concentration was 0.776 mM; Alizarin + Al 3+ +CIP represents alizarin / Al prepared in Example 1. 3+ A mixed solution of a base-ratio fluorescent probe (solution) and 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 is... 3+ The final concentration was 0.776 mM.
[0031] Figure 6 Alizarin / Al was shown in Example 2. 3+ Fluorescence emission spectrum of the base ratio fluorescent probe after adding CIP.
[0032] Figure 7 The ratio of CIP concentration to fluorescence intensity in Example 2 is shown (I 436 / I 650 The linear correlation curve of ). Detailed Implementation
[0033] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the present invention is not limited to the following embodiments.
[0034] The reagents and instruments used in the following examples are:
[0035] Aluminum trichloride hexahydrate (AlCl3) 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-hydroxyethylpiperazine ethanesulfonic acid (HEPES) and dimethyl sulfoxide were purchased from Shanghai Aladdin Chemical Reagent Co., Ltd., China; ultrapure water was used for solution preparation in the experiment; the HEPES buffer was 0.01 M HEPES, pH=5.0. All the above chemical reagents were of analytical grade, and no further purification was performed unless otherwise specified.
[0036] FL 6500 fluorescence spectrophotometer (PerkinElmer Instruments, Inc., USA); ML-3002T / 02 precision electronic balance (accuracy 0.0001 g, Mettler Toledo International Ltd.); Zeta potentiometer (Zeta, ZSU3100, UK); DF-101S constant temperature water bath (Gongyi Yuhua Instrument Co., Ltd.); KQ-250DE ultrasonic multi-frequency cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); high-speed refrigerated centrifuge (JW-3021HR).
[0037] Example 1: Alizarin / Al 3+ Construction and characterization of ratiometric fluorescent probes
[0038] Alizarin solution (500 μL, 0.5 mg / mL) and AlCl3 solution (600 μL, 4.0 mM) were thoroughly 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 above mixed solution, and the mixture was incubated at 60 °C for 15 min. Then, 500 μL of water was added to obtain the alizarin / AlCl3 solution. 3+ Base ratio fluorescent probe (solution).
[0039] from Figure 1 It can be seen that the alizarin solution exhibits a weak fluorescence signal at 650 nm under an excitation wavelength of 360 nm. With Al... 3+ The addition of alizarin significantly enhanced the fluorescence signal of the alizarin solution at 650 nm, i.e., alizarin / Al 3+ The ratiometric fluorescent probe (solution) exhibits significant red fluorescence at 650 nm and also displays significant red fluorescence under UV light. This may be due to the presence of Al. 3+ Alizarin exhibits aggregation-induced fluorescence (AIE) by coordinating with the -OH groups on its surface, thus producing strong fluorescence. The CIP solution displays intrinsic fluorescence at 436 nm, and when added to alizarin / Al... 3+ When Al is in a ratio fluorescent probe solution, 3+ The fluorescence signal of CIP was significantly enhanced, while the 650 nm fluorescence emission peak derived from alizarin remained essentially unchanged, making ratiometric fluorescence detection of CIP possible. Correspondingly, solution images under a 365 nm UV lamp further confirmed this color change.
[0040] Figure 3 For the hydrodynamic diameter of alizarin alone and the introduction of Al 3+ The hydrodynamic diameter of alizarin after the reaction can be seen from Al 3+ The introduction of [a specific ingredient] induces aggregation in the alizarin solution, changing its average diameter from 25.95 nm to 48.99 nm. Therefore, Al can be added to the alizarin solution. 3+ A fluorescent probe was constructed, and CIP was detected by calculating the ratio of fluorescence signals at 436 nm and 650 nm.
[0041] Example 2 Alizarin / Al 3+ Method for detecting ciprofloxacin using a ratio fluorescent probe
[0042] 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 prepared in Example 1. 3+After incubation at 60 °C for 15 minutes in a ratiometric fluorescent probe (solution), fluorescence spectra were recorded at room temperature under an excitation wavelength of 360 nm. Fluorescence intensities at 436 nm and 650 nm (labeled as I0.05) were measured. 436 and I 650 The slits for both excitation and emission wavelengths are 5 nm.
[0043] Depend on Figure 4 It can be seen that Al 3+ By complexing with the carboxyl and pyridone groups in CIP, the fluorescence signal of CIP is enhanced by inhibiting excited-state intramolecular proton transfer (ESIPT). It can be inferred that this is based on alizarin / Al 3+ The response mechanism of the base-ratio fluorescent probe to CIP may be due to Al 3+ After binding with alizarin, it further forms a complex with CIP, inhibiting CIP's ESIPT and leading to enhanced fluorescence signal. To verify this hypothesis, its hydrodynamic diameter was also measured, such as... Figure 5 As shown, the addition of CIP makes alizarin / Al 3+ The average diameter of the ratiometric fluorescent probe increased from 48.99 nm to 88.26 nm, indicating that CIP can interact with alizarin / Al through electrostatic interactions. 3+ Base ratio fluorescent probe binding.
[0044] The fluorescence intensity of the two fluorescence signals (I) 436 / I 650 Using the ratio as the ordinate and CIP concentration as the abscissa, a standard curve is fitted, and the linear fitting result is as follows: Figure 7 As shown, the fitted equation is y = 1.760x - 0.137 (R²). 2 =0.991, S / N=3), linear range between 0.01-10.00 μM, detection limit of 6.9 nM, indicating that the alizarin / Al of this invention... 3+ The fluorescence signal of the ratiometric fluorescent probe shows a good linear relationship with the CIP concentration, and it can be used to detect the concentration of ciprofloxacin in the sample.
[0045] Example 3 Alizarin / Al 3+ A method for detecting ciprofloxacin residues in eggs using a ratio-based fluorescent probe.
[0046] To evaluate this alizarin / Al 3+To investigate the application potential and feasibility of ratiometric fluorescent probes in practical detection, a spiked recovery method was used to analyze the residual amount of fluoroquinolones in eggs. 1.0 g of fresh egg liquid was weighed and vortexed until homogeneous. Then, 2 mL of CIP solution was added (added in triplicate, with CIP solution concentrations of 0, 0.5, 1.5, and 2.5 μM, respectively), followed by 5 mL of acetonitrile solution (acetonitrile:water, 9:1, v / v). The mixture was vortexed again until homogeneous, sonicated for 10 min, and centrifuged at 4℃ for 10 min (12000 rpm). The supernatant was collected. This process was repeated three times, and the supernatant collected was the egg extract, which was used as the test sample for detecting ciprofloxacin residues in eggs.
[0047] Alizarin / Al prepared in Example 1 3+ Add 500 μL of the above egg extract (CIP concentrations of 0, 0.5, 1.5, and 2.5 μM) to the ratiometric fluorescent probe (solution), incubate at 60°C for 15 min, and measure the fluorescence intensity at 436 nm and 650 nm at an excitation wavelength of 360 nm. Substitute the values into the standard curve in Example 2 to calculate the ciprofloxacin concentration in the egg extract. The experiment was conducted in triplicate, and the average value was taken.
[0048] The test results are shown in Table 1. The recovery rate of CIP in actual egg samples was 82.00%~102.00%, with a relative standard deviation ranging from 0.06% to 0.17%, showing good recovery rate and relative standard deviation, indicating that the alizarin / Al-based solution provided by this invention... 3+ The method of detecting ciprofloxacin residues in eggs using a ratio-based fluorescent probe has shown good performance in FQs detection and can be used for sensitive and accurate detection in CIP, with broad application prospects.
[0049] Table 1. Determination of CIP in egg samples
[0050]
[0051] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.
Claims
1. Alizarin 3+ The application of a ratiometric fluorescent probe in the detection of ciprofloxacin is characterized by, The alizarin / Al 3+ The alizarin / AlCl3 ratio fluorescent probe is prepared by mixing alizarin solution, AlCl3 solution, and buffer solution, using water as a solvent; the alizarin / AlCl3 ratio fluorescent probe is prepared by mixing alizarin solution, AlCl3 solution, and buffer solution, using water as a solvent; 3+ The ratiometric fluorescent probe, when excited by excitation light at a wavelength of 355-365 nm in the presence of ciprofloxacin, exhibits red fluorescence at 647-653 nm and blue fluorescence at 433-439 nm. The ratio of the fluorescence intensity of red and blue fluorescence has a linear relationship with the concentration of ciprofloxacin, which can be used to achieve quantitative detection of ciprofloxacin.
2. Alizarin 3+ The application of a ratiometric fluorescent probe in the detection of ciprofloxacin is characterized by, The alizarin / Al 3+ The alizarin / AlCl3 ratio fluorescent probe is prepared by mixing alizarin solution, AlCl3 solution, and buffer solution, using water as a solvent; the alizarin / AlCl3 ratio fluorescent probe is prepared by mixing alizarin solution, AlCl3 solution, and buffer solution, using water as a solvent; 3+ The ratiometric fluorescent probe, in the presence of ciprofloxacin, exhibits red fluorescence at 647-653 nm and blue fluorescence at 433-439 nm upon excitation with light at a wavelength of 355-365 nm; the alizarin / Al 3+ The ratiometric fluorescent probe changes color from red through purple to blue as the concentration of ciprofloxacin increases, enabling semi-quantitative visual detection of ciprofloxacin.
3. The alizarin / Al according to claim 1 or 2 3+ The application of a ratiometric fluorescent probe in the detection of ciprofloxacin is characterized by, The alizarin / Al 3+ The preparation method of the base ratio fluorescent probe includes the following steps: (1) Prepare an alizarin solution with a concentration of 0.4-0.6 mg / mL using water as a solvent; (2) Prepare an AlCl3 solution with a concentration of 3-5 mM using water as a solvent; (3) Mix the alizarin solution and AlCl3 solution thoroughly at a volume ratio of (0.7-1):1, and let stand at room temperature to obtain a mixed solution; (4) Add buffer solution or buffer solution and water to the mixed solution obtained in step (3), and incubate at 55-65 °C for 10-20 minutes to obtain alizarin / Al. 3+ Base ratio fluorescent probe; the alizarin / Al 3+ The ratiometric fluorescent probe is a solution in which the final concentration of alizarin is in the range of 0.05-0.10 mg / mL. 3+ The final concentration is in the range of 0.5-1.0 mM.
4. The alizarin / Al as described in claim 3 3+ The application of a ratiometric fluorescent probe in the detection of ciprofloxacin is characterized by, The buffer solution used is HEPES buffer with a concentration of 0.005-0.015 M and a pH of 4.8-5.
2.
5. The alizarin / Al as described in claim 3 3+ The application of a ratiometric fluorescent probe in the detection of ciprofloxacin is characterized by, In step (4), the volume ratio of the mixed solution to the buffer solution is in the range of (0.6-0.9):1; the volume ratio of the mixed solution to water is in the range of (1.8-2.7):
1.
6. The alizarin / Al according to claim 1 or 2 3+ The application of a ratiometric fluorescent probe in the detection of ciprofloxacin is characterized by, The specific application method includes the following steps: 1) Using water as a solvent, prepare ciprofloxacin standard solutions of different concentrations, ranging from 0 to 15 μM; 2) The ciprofloxacin standard solutions of different concentrations described in step 1) are added in parallel to the alizarin / Al solution of the present invention. 3+ In the ratiometric fluorescent probe, after incubation at 55-65 ℃ for 10-20 minutes, the fluorescence intensity ratio at 433-439 nm and 647-653 nm was measured at an excitation wavelength of 355-365 nm. A standard curve was established between the fluorescence intensity ratio and the concentration of ciprofloxacin standard solution, with the fluorescence intensity ratio as the ordinate and the concentration of ciprofloxacin standard solution as the abscissa. 3) Under the same conditions as in step 2), measure the fluorescence intensity ratio at 433-439 nm and 647-653 nm of the sample to be tested, and then obtain the concentration of ciprofloxacin in the sample to be tested based on the standard curve obtained in step 2).
7. The alizarin / Al as described in claim 6 3+ The application of a ratiometric fluorescent probe in the detection of ciprofloxacin is characterized by, The sample to be tested is an egg extract, which is prepared as follows: fresh egg liquid is vortexed and mixed evenly, then ultrasonically treated with a mixture of acetonitrile and water, and then the supernatant is collected by centrifugation, which is the egg extract and is used as the sample to detect ciprofloxacin residues in eggs.
8. The alizarin / Al as described in claim 7 3+ The application of a ratiometric fluorescent probe in the detection of ciprofloxacin is characterized by, The egg extract was diluted with HEPES buffer and used as the test sample for detecting ciprofloxacin residues in eggs.
9. A method based on alizarin / Al 3+ A method for detecting ciprofloxacin residues in eggs using a ratio-based fluorescent probe, characterized in that... The specific steps are as follows: S1, Preparation of Alizarin / Al 3+ Alizarin / AlCl3 ratio fluorescent probe: Mix 0.4-0.6 mg / mL alizarin aqueous solution and 3-5 mM AlCl3 aqueous solution at a volume ratio of (0.7-1):1 to obtain a mixed solution; mix the resulting mixed solution with buffer, or mix the resulting mixed solution with buffer and water, and then incubate at 55-65 ℃ for 10-20 minutes to obtain alizarin / AlCl3 ratio fluorescent probe. 3+ A ratiometric fluorescent probe, wherein the final concentration of alizarin is in the range of 0.05-0.10 mg / mL, Al 3+ The final concentration is in the range of 0.5-1.0 mM; S2. Add ciprofloxacin standard solutions of different concentrations to the alizarin / Al obtained in step S1 in parallel. 3+ After incubating the ratiometric fluorescent probe at 55-65 ℃ for 10-20 minutes, the fluorescence intensity ratio at 433-439 nm and 647-653 nm was measured at an excitation wavelength of 355-365 nm. A standard curve was established between the fluorescence intensity ratio and the concentration of ciprofloxacin standard solution, with the fluorescence intensity ratio as the ordinate and the concentration of ciprofloxacin standard solution as the abscissa. S3. Using egg extract from fresh eggs as the test sample for detecting ciprofloxacin residue in eggs, the fluorescence intensity ratio at 433-439 nm and 647-653 nm is measured under the same conditions as in step S2. Then, the concentration of ciprofloxacin in the test sample is obtained according to the standard curve obtained in step S2.
10. A method based on alizarin / Al according to claim 9 3+ A method for detecting ciprofloxacin residues in eggs using a ratio-based fluorescent probe, characterized in that... The linear range of the standard curve is within the concentration range of ciprofloxacin from 0.1 to 15 μM.