Luminol-Eu-NCDs ratiometric fluorescent probe as well as preparation method and application thereof

Abstract

The invention belongs to the field of antibiotic detection, and relates to detection of norfloxacin (NOR). The invention provides a Luminol-Eu-NCDs ratiometric fluorescent probe as well as a preparation method and application of the Luminol-Eu-NCDs ratiometric fluorescent probe. According to the application, a novel ratiometric fluorescence sensor is constructed through covalent linkage of Luminol-Eu self-assembled and synthesized by AIE and NCDs derived from Listeria monocytogenes. The Luminol-Eu NPs with the AIE effect overcomes the fluorescence quenching characteristic of a traditional fluorophore in a high concentration or aggregation state, so that the reference fluorescence brightness of fluorescence analysis is greatly improved. In addition, due to the introduction of the Luminol-Eu NPs, the sensitivity and the accuracy of NOR detection can also be improved. On the basis, a simple ratio fluorescence sensor with high selectivity is developed, and the ratio fluorescence sensor is used for detecting NOR residues in a milk sample.

Description

Technical Field

[0001] The present invention belongs to the field of antibiotic detection and relates to the detection of norfloxacin (NOR). Background Art

[0002] Norfloxacin (NOR) is a class of chemically synthesized broad-spectrum antibiotics. Due to its high antibacterial activity, it is widely used in animal husbandry and aquaculture for the prevention and treatment of bacterial infections. However, due to unreasonable use (such as over-addition or non-compliance with the withdrawal period) and its poor biodegradability, these antibiotics are likely to remain in food animals and enter the human body through the food chain. Long-term intake of low-dose antibiotic residues may not only lead to imbalance of the human intestinal flora, but also accelerate the generation of bacterial drug resistance, and even pose potential hazards to organs such as the immune system, liver and kidneys. In addition, antibiotic residues may further spread through environmental media (such as water bodies, soil), exacerbate ecological environmental pollution, form a drug resistance gene pool, and pose a double threat to public health and the ecosystem. Therefore, the determination of norfloxacin residues in food is of great significance for ensuring food safety and maintaining public health.

[0003] At present, a variety of analytical methods have been developed for the accurate quantification of NOR in food. Instrumental analytical methods include micellar electrokinetic chromatography, high performance liquid chromatography (HPLC), and high performance liquid chromatography-tandem mass spectrometry, etc. These methods have been widely recognized for their high sensitivity, high accuracy and low detection limit. However, they also have some limitations, such as cumbersome operation, long detection time, requiring professional training, and high equipment cost. At present, new analytical techniques for detecting NOR emerge in an endless stream, such as electrochemical detection method, colorimetric method, fluorescence detection method, etc. Among them, the fluorescence detection method has the characteristics of low cost, high sensitivity, simple operation, fast reaction speed and visualization.

[0004] Fluorescent probes are important tools for fluorescence analysis. So far, a variety of materials have been widely used to prepare probes for fluorescence sensors, including coordination polymer nanoparticles, carbon dots (CDs), and metal-organic framework (MOF) materials. Coordination polymers with aggregation-induced emission (AIE) effects can overcome the fluorescence quenching effect of traditional fluorophores at high concentrations or in the aggregated state, thus providing a strong fluorescence reference signal for visual fluorescence analysis. For example, Yao et al. synthesized a Luminol-Eu coordination polymer for the sensitive and selective detection of tetracycline in milk. Qi et al. synthesized Luminol-terbium(III) coordination nanoparticles for the quantitative detection of thrombin in serum. CDs are a new type of fluorescent material with excellent optical stability, strong FL characteristics, surface functionalization, and biocompatibility, and have the advantages of easy availability of raw materials, simple synthesis, and easy functionalization. These superior properties make CDs have broad application prospects in the fields of environmental monitoring, biomedicine, and chemical sensing. In addition, it has been reported that nitrogen doping can endow CDs with new structures and photoluminescence properties, and nitrogen-doped carbon dots (NCDs) have been used in the field of fluorescence sensors. For example, Zhang et al. synthesized highly fluorescent NCDs by pyrolyzing disodium ethylenediaminetetraacetate for the quantitative detection of glyphosate. Wang et al. prepared NCDs by a hydrothermal method for the determination of chlorpyrifos in fruit juice. However, the single wavelength of the energy of the fluorescent probe depends on the enhancement or attenuation of a single signal and is easily interfered by non-analytical factors (such as the pH value of the medium, instrument deviation, and polarity), lamp tube bleaching, etc., resulting in "false positive errors". Compared with single-wavelength fluorescent probes, the ratio fluorescence detection method containing two or more different wavelength emission bands has more superior adaptability and stronger anti-interference ability. Therefore, the ratio fluorescence detection method can effectively eliminate or reduce color differences through self-calibration, thus having higher sensitivity and accuracy. Summary of the Invention

[0005] To solve the above technical problems, the present invention proposes a Luminol-Eu-NCDs ratio fluorescent probe and its preparation method and application.

[0006] The technical solution of the present invention is realized as follows: In the first aspect, the present application provides a preparation method of a Luminol-Eu-NCDs ratio fluorescent probe, and the steps are as follows: (1) Dissolve luminol and Eu(NO 3 ). 3 ·6H 2 O in Tris-HCl buffer solution, and prepare Luminol-Eu through magnetic stirring reaction; (2) Using Listeria monocytogenes and urea as precursors and N,N-dimethylacetamide as the reaction medium, synthesize NCDs by a solvothermal method; (3) Mix and stir the Luminol-Eu from step (1) and the NCDs from step (2) in a solvent, centrifuge, and discard the supernatant to obtain the Luminol-Eu-NCDs ratiometric fluorescence probe.

[0007] In the above step (1), the molar ratio of luminol to Eu(NO 3 ) 3 ·6H 2 O is 1:6 - 10; the pH of the Tris-HCl buffer solution is 6 - 10. The temperature of the above magnetic stirring is 20 - 40 °C and the time is 20 - 50 min.

[0008] In the above step (3), the mass ratio of Luminol-Eu to NCDs is 280 - 320:1; the solvent is water.

[0009] In the above step (3), the temperature of the mixing and stirring is 20 - 40 °C and the time is 20 - 50 min.

[0010] Specifically: First, add 2.5 mL of 10 mmol / L luminol, 2 mL of 100 mmol / L Eu(NO 3 ) 3 ·6H 2 O and 2 mL of Tris-HCl buffer solution with pH = 8 into a beaker in sequence and mix well. Then place the mixture on a magnetic stirrer and continuously stir at 30 °C for 30 minutes to obtain the Luminol-Eu ratiometric fluorescence probe.

[0011] Then, culture Listeria monocytogenes to a concentration of 10 8 -10 9 CFU / mL, centrifuge and wash to obtain the Listeria monocytogenes precipitate; add urea to the obtained Listeria monocytogenes precipitate, and then resuspend it in N, N-dimethylacetamide to obtain a suspension; react the suspension at 180 °C for 10 h to obtain a dark brown solution, filter it through a 0.22 μm filter membrane by centrifugation, purify, and rotary evaporate to obtain NCDs.

[0012] Finally, stir a mixture with a mass ratio of Luminol-Eu to NCDs of 300:1 at 30 °C for 30 min. Centrifuge the obtained mixed solution at 6500 g for 5 min and discard the supernatant. Then, add ultrapure water and stir evenly. Finally, place the Luminol-Eu-NCDs ratiometric fluorescence probe solution at -4 °C.

[0013] In a second aspect, there is provided a Luminol-Eu-NCDs ratiometric fluorescence probe prepared by using the above method, having an excitation wavelength of 270 nm and two typical emission peaks at 430 nm and 542 nm.

[0014] In a third aspect, the present application provides the use of the above Luminol-Eu-NCDs ratiometric fluorescence probe in the detection of norfloxacin for non-disease diagnosis purposes.

[0015] In a third aspect, the present application provides a method for detecting norfloxacin for non-disease diagnosis purposes. The steps are as follows: The Luminol-Eu-NCDs ratiometric fluorescence probe solution and the sample to be detected are sequentially added to a cuvette in an equal volume ratio. After stabilization, the fluorescence intensity ratio at 542 nm and 430 nm is recorded as y, and is substituted into the linear equation y = -0.0568x + 6.73812 to calculate the concentration x of norfloxacin in the sample to be detected.

[0016] The concentration of the above Luminol-Eu-NCDs ratiometric fluorescence probe solution is 4 - 8 mg / mL; the sample to be detected is a pretreated food.

[0017] Furthermore, the food is a fluid food. For example: milk, soy milk, fruit juice, beer, etc.

[0018] The present invention has the following beneficial effects: 1. In the present application, through the covalent connection of Luminol-Eu synthesized by AIE self-assembly and NCDs derived from Listeria monocytogenes, a novel ratiometric fluorescence sensor is constructed. First, Luminol-Eu nanoparticles (Luminol-Eu NPs) are synthesized by a self-assembly method based on the AIE strategy. Then, NCDs are synthesized by using Listeria monocytogenes and urea through a simple and green method. Finally, the Luminol-Eu-NCDs ratiometric fluorescence sensor is synthesized by the covalent connection method of Luminol-Eu NPs and NCDs. Luminol-Eu NPs with AIE effect overcome the fluorescence quenching characteristics of traditional fluorophores at high concentrations or in an aggregated state, thus greatly improving the reference fluorescence brightness of fluorescence analysis. In addition, the introduction of Luminol-Eu NPs can also improve the sensitivity and accuracy of NOR detection, as Figure 1 shown. When the Luminol-Eu-NCDs solution reacts with NOR, the fluorescence intensity at the wavelength of 542 nm will be greatly quenched, while the fluorescence intensity at the wavelength of 430 nm is slightly quenched.

[0019] 2. The ratiometric fluorescent probe Luminol-Eu-NCDs of the present application can provide significant robustness, sensitivity, and selectivity for this method through dual-signal response readout. In addition, the ratiometric method uses the fluorescence intensity at 430 nm as the internal reference and the fluorescence intensity at 542 nm as the detection signal, effectively reducing the influence of environmental fluctuations and enhancing its applicability in practical applications. The ratiometric fluorescent sensor based on Luminol-Eu-NCDs has a rapid fluorescence quenching response, a wide detection range, and a fast response time, and can detect NOR with acceptable selectivity and sensitivity. The ratiometric fluorescent sensor based on Luminol-Eu-NCDs can also quantitatively detect NOR in milk samples with satisfactory recoveries. This patent has established a reliable, rapid, and accurate method for determining NOR residues in foods and the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following-described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 Schematic diagram for the preparation of Luminol-Eu-NCDs and NOR detection.

[0022] Figure 2 Characterization and spectra of Luminol-Eu-NCDs; where (a) SEM image of Luminol-Eu-NCDs; (b) FT-IR spectra of NCDs, Luminol-Eu, and Luminol-Eu-NCDs; (c) full-scale XPS measurement spectrum of Luminol-Eu-NCDs; high-resolution XPS spectra of C1s (d), O1s (e), N1s (f), and Eu3d (g) of Luminol-Eu-NCDs; (h) UV-vis spectra of NOR, Luminol-Eu-NCDs, and Luminol-Eu-NCDs with NOR; (i) photoluminescence spectrum of Luminol-Eu-NCDs.

[0023] Figure 3 XRD pattern of Luminol-Eu-NCDs.

[0024] Figure 4Fluorescence mechanism of Luminol-Eu-NCDs; among which (a) Fluorescence spectra of Luminol-Eu-NCDs at different excitation wavelengths; (b) Influence of NaCl concentration on the fluorescence intensity ratio of the Luminol-Eu-NCDs ratiometric fluorescence sensor at 542 nm and 430 nm; (c) Different fluorescence peak intensities of Luminol-Eu-NCDs at 542 nm and 430 nm under different pH conditions; (d) Variation of the fluorescence intensity ratio of Luminol-Eu-NCDs at 542 nm and 430 nm when pH is 2 - 10; (e) Photostability of Luminol-Eu-NCDs under irradiation of a xenon lamp with an excitation wavelength of 542 nm; (f) Fluorescence stability of Luminol-Eu-NCDs without NOR.

[0025] Figure 5 Feasibility of detecting NOR by Luminol-Eu-NCDs; among which (a) Ultraviolet-visible absorption spectrum of NOR and the excitation wavelength of Luminol-Eu-NCDs at 542 nm; (b) Fluorescence decay curve of Luminol-Eu-NCDs at 542 nm after introducing NOR; (c) Stern-Volmer plot of the quenching of Luminol-Eu-NCDs by NOR.

[0026] Figure 6 Sensitivity and specificity of detecting NOR by Luminol-Eu-NCDs; among which (a) Fluorescence spectra of Luminol-Eu-NCDs with different NOR concentrations (λex = 270 nm); (b) Relationship between F542 / F430 and NOR concentration; (c) Color parameters of Luminol-Eu-NCDs after adding different concentrations of NOR on the CIE diagram; (d) Specificity test of detecting NOR by Luminol-Eu-NCDs. Specific implementation mode

[0027] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0028] The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0029] Example 1 Preparation of the Luminol-Eu-NCDs ratiometric fluorescence probe in this example is carried out in the following steps: (1)Add 2.5 mL of 10 mmol / L luminol, 2 mL of 100 mmol / L Eu(NO 3 ) 3 ·6H 2 O and 2 mL of Tris-HCl buffer with pH = 8 into a beaker in sequence and mix well. Then place the mixture on a magnetic stirrer and continuously stir at 30 °C for 30 minutes.

[0030] (2)Listeria monocytogenes is shaken well in 30 mL of TSB medium and cultured overnight at 37 °C. Collect 10 9 CFU / mL cells, centrifuge at 4000 × g for 3 min, and wash 3 times with ultrapure water. Then, add 10 g of urea to the obtained cell precipitate, and resuspend it in 20 mL of N, N-dimethylacetamide. Mix the solution evenly, transfer it to a 150 mL Teflon-lined stainless steel autoclave, react at 180 °C for 10 h, centrifuge the resulting dark brown solution at 8000 × g for 10 min, and filter the supernatant through a 0.22 μm filter membrane. Then, use silica gel column chromatography to purify the product with methanol and ethyl acetate as eluents. The collected eluate is dried with a vacuum rotary evaporator, diluted with water, and stored at 4 °C for later use.

[0031] (3)Dissolve the mixture with a mass ratio of Luminol-Eu to NCDs of 300:1 in ultrapure water and stir at 30 °C for 30 min. Centrifuge the obtained mixed solution at 6500 g for 5 min and discard the supernatant. Then, add ultrapure water and stir evenly. Finally, place the Luminol-Eu-NCDs ratiometric fluorescence probe solution at -4 °C.

[0032] The morphology of Luminol-Eu-NCDs was characterized by scanning electron microscopy (SEM). As Figure 2 shown in a, the prepared material presents rectangular flakes with a certain thickness, clear contour shape, uniform individual distribution, and good particle dispersion. The average length of Luminol-Eu-NCDs is 15 μm, and the average width is 8 μm. It is preliminarily confirmed that Luminol-Eu-NCDs are successfully synthesized. The crystal structure of Luminol-Eu-NCDs was observed by X-ray diffraction (XRD). The XRD pattern ( Figure 3 )shows a characteristic diffraction peak at 20.9° corresponding to the (202) crystal plane, thus inferring that Luminol-Eu-NCDs have good crystallinity.

[0033] To further prove the successful synthesis of Luminol-Eu-NCDs, Fourier transform infrared (FT-IR) spectra of NCDs, Luminol-Eu, and Luminol-Eu-NCDs were used to determine the elemental composition and chemical state of the synthesized Luminol-Eu-NCDs. As Figure 2 shown in -1 b, it can be clearly seen from the FT-IR spectrum of NCDs that the peak at 3700 - 3000 cm -1 is due to the O-H stretching vibration, and the region at 1870 - 1540 cm -1 is for the C=O and C=C stretching vibrations. For Luminol-Eu, the absorption peaks at 2974 cm -1 , 2887 cm -1 , 2360 cm -1 , and 1658 cm -1 are attributed to C-H stretching vibration, CHO stretching vibration, N=C=O stretching vibration, and C=O stretching vibration, respectively. Luminol-Eu-NCDs contain some characteristic peaks of NCDs and Luminol-Eu, but the peak shape at 2360 cm -1 shows a slight change. In addition, the stretching vibrations of CHO and C-H shift from 2974 cm -1 and 2887 cm -1 to 2976 cm -1 and 2889 cm -1 , respectively. All these results indicate the successful preparation of Luminol-Eu-NCDs. In addition, X-ray photoelectron spectroscopy (XPS) analysis ( Figure 2 c) further proves the successful synthesis of Luminol-Eu-NCDs through the positions and intensities of the characteristic peaks observed in the C1s, O1s, N1s, and Eu3d spectra. In Figure 2 d, the C1s peaks at 284.7 eV, 286.4 eV, and 289.3 eV are attributed to C-C / C=C, C-O, and C=O, respectively. As Figure 2 shown in Figure 2 e, the peaks observed at 530.7 eV, 531.5 eV, and 532.5 eV correspond to Eu-O, C=O, and C-O, respectively. As Figure 2 shown in Figure 2 f, the high-resolution spectrum of N1s has three peaks at 399.7 eV, 402.7 eV, and 407.1 eV, which are attributed to N-H, N-C, and N-O, respectively. The high-resolution Eu 3d spectrum ( Figure 2 g) has two typical peaks at 1155.7 eV and 1164.3 eV, belonging to Eu3d 3 / 2 , and one peak at 1134.6 eV belonging to Eu3d 5 / 2Typical peaks. The results showed that Luminol-Eu-NCDs were synthesized through covalent linkage between Luminol-Eu NPs and NCDs, and a fluorescent nanoprobe of Luminol-Eu-NCDs was successfully prepared.

[0034] The optical properties of the Luminol-Eu-NCDs ratiometric fluorescent probe were studied using ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy. The ultraviolet-visible absorption peaks of NOR were located at 273 and 323 nm ( Figure 2 h), while Luminol-Eu-NCDs had three peaks at 210, 300, and 350 nm. Among them, the absorption peak at 210 nm was mainly due to the π-π* transition of C-C, while the absorption peaks at 300 nm and 350 nm were attributed to the n-π* transitions of -NH 2 and -C=O groups on the benzene ring. The reaction system of Luminol-Eu-NCDs and NOR showed the characteristic absorption peaks of Luminol-Eu-NCDs and NOR, and the peak positions shifted slightly. In the fluorescence spectrum ( Figure 2 i), it could be observed that the excitation wavelength of Luminol-Eu-NCDs was 270 nm, and there were two typical emission peaks at 430 nm and 542 nm.

[0035] Example 2 The preparation steps of the Luminol-Eu-NCDs ratiometric fluorescent probe in this example were as follows: (1) 2.5 mL of 10 mmol / L luminol, 1.5 mL of 100 mmol / L Eu(NO 3 ) 3 ·6H 2 O and 2 mL of Tris-HCl buffer solution with pH = 7 were successively added to a beaker and mixed well. Then the mixture was placed on a magnetic stirrer and continuously stirred at 20 °C for 35 minutes.

[0036] (2) Listeria monocytogenes was shaken well in 30 mL of TSB medium and cultured overnight at 37 °C. 10 9CFU / mL cells were centrifuged at 4000 × g for 3 min and washed three times with ultrapure water. Then, 10 g of urea was added to the obtained cell pellet, and it was resuspended in 20 mL of N, N-dimethylacetamide. The solution was mixed evenly, transferred to a 150 mL Teflon-lined stainless-steel autoclave, and reacted at 160 °C for 8 h. The resulting dark brown solution was centrifuged at 8000 × g for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. Then, using methanol and ethyl acetate as eluents, the product was purified by silica gel column chromatography. The collected eluate was dried with a vacuum rotary evaporator, diluted with water, and stored at 4 °C for later use.

[0037] (3)A mixture with a mass ratio of Luminol-Eu to NCDs of 320:1 was dissolved in ultrapure water and stirred at 30 °C for 30 min. The resulting mixed solution was centrifuged at 6500 g for 5 min, and the supernatant was discarded. Then, ultrapure water was added and stirred evenly. Finally, the Luminol-Eu-NCDs ratio fluorescence probe solution was placed at -4 °C.

[0038] Example 3 The preparation steps of the Luminol-Eu-NCDs ratio fluorescence probe in this example are as follows: (1)2.5 mL of 10 mmol / L luminol, 2.5 mL of 100 mmol / L Eu(NO 3 ) 3 ·6H 2 O and 2 mL of Tris-HCl buffer with a pH of 10 were successively added to a beaker and mixed thoroughly. Then the mixture was placed on a magnetic stirrer and continuously stirred at 40 °C for 20 minutes.

[0039] (2)Listeria monocytogenes was shaken well in 30 mL of TSB medium and cultured overnight at 37 °C. 10 9 CFU / mL cells were centrifuged at 4000 × g for 3 min and washed three times with ultrapure water. Then, 10 g of urea was added to the obtained cell pellet, and it was resuspended in 20 mL of N, N-dimethylacetamide. The solution was mixed evenly, transferred to a 150 mL Teflon-lined stainless-steel autoclave, and reacted at 200 °C for 10 h. The resulting dark brown solution was centrifuged at 8000 × g for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. Then, using methanol and ethyl acetate as eluents, the product was purified by silica gel column chromatography. The collected eluate was dried with a vacuum rotary evaporator, diluted with water, and stored at 4 °C for later use.

[0040] (3) Stir the mixture with a mass ratio of Luminol-Eu to NCDs of 280:1 at 20 °C for 50 min. Centrifuge the resulting mixed solution at 6500 g for 5 min and discard the supernatant. Then, add ultrapure water and stir evenly. Finally, place the Luminol-Eu-NCDs ratio fluorescence probe solution at -4 °C.

[0041] Example 4 The preparation steps of the Luminol-Eu-NCDs ratio fluorescence probe in this example are as follows: (1) Add 2.5 mL of 10 mmol / L luminol, 2.25 mL of 100 mmol / L Eu(NO 3 ) 3 ·6H 2 O and 2 mL of pH = 9 Tris-HCl buffer solution into a beaker in sequence and mix well. Then place the mixture on a magnetic stirrer and continuously stir at 40 °C for 20 minutes.

[0042] (2) Shake Listeria monocytogenes in 30 mL of TSB medium and culture overnight at 37 °C. Collect 10 9 CFU / mL cells, centrifuge at 4000 × g for 3 min, and wash 3 times with ultrapure water. Then, add the obtained cell precipitate to 10 g of urea and resuspend in 20 mL of N, N-dimethylacetamide. Mix the solution evenly, transfer it to a 150 mL Teflon-lined stainless steel autoclave, react at 190 °C for 10 h, centrifuge the resulting dark brown solution at 8000 × g for 10 min, and filter the supernatant through a 0.22 μm filter membrane. Then, use silica gel column chromatography to purify the product with methanol and ethyl acetate as eluents. The collected eluate is dried with a vacuum rotary evaporator, diluted with water, and stored at 4 °C for standby.

[0043] (3) Stir the mixture with a mass ratio of Luminol-Eu to NCDs of 290:1 at 35 °C for 45 min. Centrifuge the resulting mixed solution at 6500 g for 5 min and discard the supernatant. Then, add ultrapure water and stir evenly. Finally, place the Luminol-Eu-NCDs ratio fluorescence probe solution at -4 °C.

[0044] Analysis of implementation effects Accurate detection of analytes is crucial for analytical methods. However, the sensitivity of the response can be affected by various factors. The excitation wavelength and response time are key factors affecting the detection sensitivity of analytical methods. In this study, the detection conditions were optimized to improve the performance of the developed analyte detection probe, such as excitation wavelength, ionic strength, pH value, photobleaching resistance, and fluorescence intensity stability. These optimizations help improve the accuracy and precision of the analytical method. As Figure 4 shown in a, we evaluated the fluorescence intensity of Luminol-Eu-NCDs at different excitation wavelengths. It can be clearly seen that when the excitation wavelength varies in the range of 250 - 320 nm, the fluorescence emission intensity of NCDs in Luminol-Eu-NCDs first increases and then decreases, and there is a red shift phenomenon. When excited at 270 nm, the maximum emission intensity is 542 nm. At the same time, when the excitation wavelength changes from 250 nm to 320 nm, the fluorescence intensity at the reference signal of 430 nm also first increases and then decreases. The fluorescence intensity at 430 nm reaches the maximum at the excitation wavelength of 290 nm. Considering the fluorescence at 430 nm as the reference signal, we selected an excitation wavelength of 270 nm for subsequent analysis.

[0045] By changing the NaCl concentration, the effect of ionic strength on the fluorescent probe was systematically studied. As Figure 4 shown in b, as the NaCl concentration increases, the intensity ratio of Luminol-Eu-NCDs at wavelengths of 542 nm and 430 nm always remains within a certain fluctuation range, without very obvious changes. It can be seen that this Luminol-Eu-NCDs has strong salt tolerance and can remain relatively stable even in a high-concentration salt solution environment. In addition, the effect of different pH values on this fluorescent probe was also studied. As Figure 4 shown in c and 4d, the pH value has a great influence on the detection system. Comparing Figure 4 c and Figure 4 d, it can be found that under the condition of pH = 2, the FL intensity ratio at the excitation wavelengths of 542 nm and 430 nm is the smallest, but the intensities of these two peaks are very low at this time, which does not meet the requirements of the experiment itself. Under the condition of pH = 3, the peak intensity at 430 nm is the strongest, and the peak intensity at 542 nm is also relatively high. When the excitation wavelengths are 542 nm and 430 nm, the ratio of fluorescence intensities is only second to that at pH = 2. Therefore, after comparison, the Tris-HCl buffer solution with pH = 3 was finally selected as the optimal pH value for diluted Luminol-Eu-NCDs. As Figure 4As shown in Fig. e, we also investigated the photostability of Luminol-Eu-NCDs under xenon lamp irradiation. To test the photobleaching resistance performance of Luminol-Eu-NCDs, 1 mL of the pre-prepared Luminol-Eu-NCDs solution was diluted with Tris-HCl buffer (0.02 mol / L, pH = 3) and placed in a quartz test tube. Its photobleaching resistance performance was tested at an excitation wavelength of 270 nm, an emission wavelength of 542 nm, and a scanning time of 30 min. As can be seen from Figure 4 Fig. e, under long-term xenon lamp irradiation, the fluorescence intensity of Luminol-Eu-NCDs remained basically unchanged, indicating that Luminol-Eu-NCDs had good photobleaching resistance performance. In addition, the photostability and time stability of the fluorescence probe are key factors for evaluating the performance of the sensor. Based on the above considerations, the time stability test of the Luminol-Eu-NCDs ratio fluorescence probe was carried out. The Luminol-Eu-NCDs solution was taken out from the quartz test tube, and the fluorescence intensity at the emission wavelength of 542 nm was recorded every 2 min. The experimental results are as shown in Figure 4 Fig. f. As time went by, the fluorescence intensity value always fluctuated within a certain range without obvious decrease, indicating that the Luminol-Eu-NCDs fluorescent nanoprobe had good photostability and time stability. Therefore, the Luminol-Eu-NCDs fluorescent nanoprobe had good photobleaching resistance performance and time stability, was suitable for the analysis of actual samples, and had great potential in analytical detection.

[0046] Regarding the above content, the mechanism of NOR quenching the fluorescence of Luminol-Eu-NCDs was explored. As shown in Figure 5 Fig. a, the ultraviolet-visible absorption spectrum of the NOR solution overlapped with the excitation spectrum of Luminol-Eu-NCDs, indicating that NOR might quench the fluorescence of Luminol-Eu-NCDs through the inner filter effect (IFE).

[0047] To further determine the fluorescence quenching mechanism of Luminol-Eu-NCDs, the fluorescence lifetime of Luminol-Eu-NCDs was studied. The feasibility of using Luminol-Eu-NCDs to detect NOR was further analyzed by the following experiment: Photoluminescence sensing experiments were carried out in aqueous solution at room temperature. The synthesized Luminol-Eu-NCDs stock solution was further diluted with ultrapure water at a ratio of 1:150. Diluted solutions of Luminol-Eu-NCDs with different concentrations and NOR solution were added to a quartz cuvette in equal volume ratios in sequence. After a 10-minute stabilization period, the fluorescence emission spectra were recorded five times to evaluate the measurement reproducibility. The excitation wavelength was 270 nm. The slit widths of excitation and emission were both set to 2.5 nm, and the photomultiplier tube (PMT) voltage was set to 700 V.

[0048] The results are as Figure 5 shown in Fig. b. The fluorescence lifetime of Luminol-Eu-NCDs changed significantly before and after the addition of NOR, indicating that IFE was not the cause of the fluorescence quenching of Luminol-Eu-NCDs. In addition, we speculated that the fluorescence quenching mechanism of Luminol-Eu-NCDs might be static quenching or dynamic quenching. Generally speaking, the quenching mechanism of quencher on fluorophore includes two modes: dynamic quenching and static quenching.

[0049] Due to the intermolecular interaction between the quencher and the fluorophore, static quenching occurs by forming a non-fluorescent complex. Dynamic quenching refers to the collision of the quencher with the excited-state fluorescent molecule, resulting in a decrease in fluorescence intensity. The Stern-Volmer equation was used to analyze and determine the fluorescence quenching.

[0050] Fluorescence quenching can be described by the Stern-Volmer equation (Equation 1): (1) F 0 and F are the fluorescence intensities of Luminol-Eu-NCDs without and with NOR, respectively. Cq is the concentration of NOR. k SV is the dynamic quenching constant. kq is the quenching rate constant, τ 0 is the fluorescence lifetime of Luminol-Eu-NCDs (τ 0 = 5.02 ns). F 0 / F showed a good linear relationship with the Stern-Volmer plot ( Figure 5 c). As Figure 3 shown, there was a good linear correlation between F 0 / F and the Stern-Volmer plot. The results showed that k SV was 2.46×10 -2 , kq was 4.91×10 6 M -1 s-1 , lower than the possible value of dynamic quenching effect, 1.0×10 10 M -1 s -1 . These results prove that dynamic quenching is the cause of fluorescence quenching.

[0051] Sensitivity is a key factor in evaluating fluorescence probes. The fluorescence intensity titration method was used to quantify NOR. Figure 6 a shows the change in fluorescence intensity of Luminol-Eu-NCDs under the action of different concentrations of NOR under the conditions of Luminol-Eu-NCDs concentration of 6 mg / mL and pH of 3. As Figure 6 shown in a, the obtained fluorescence spectra show that when the NOR concentration increases from 0 μmol / L to 100 μmol / L, the fluorescence intensity of Luminol-Eu-NCDs at 542 nm decreases significantly, and the decrease at 430 nm is smaller. Therefore, a ratio fluorescence method for NOR detection can be established. In Figure 6 b, in a wide concentration range of 0 - 100 μmol / L, there is a good linear response between the fluorescence intensity ratio of Luminol-Eu-NCDs at 542 nm and 430 nm and different concentrations of NOR. Its linear equation is y = -0.0568x + 6.73812, and the linear correlation coefficient is 0.98. After calculation, the detection limit (LOD) of this experiment is 68 nmol / L. The calculation of LOD is based on 3σ, where σ represents the standard deviation of blank samples. Compared with previous NOR detection methods, this ratio fluorescence probe has the same LOD level and a wider linear range. At the same time, Figure 6 c gives the CIE1931 colorimetric coordinates of Luminol-Eu-NCDs in the presence of NOR. The results show that the ratio fluorescence probe based on Luminol-Eu-NCDs can effectively detect NOR through the fluorescence color change of Luminol-Eu-NCDs.

[0052] Excellent selectivity performance is a key parameter for evaluating analytical methods. The reaction of the Luminol-Eu-NCDs ratio fluorescence probe with other antibiotics (GEN, THI, LIN, FFC, MTZ, NEO, SMZ) was studied to analyze the selectivity of this method. Interfering antibiotics such as gentamicin sulfate (GEN), thiamphenicol (THI), lincomycin (LIN), florfenicol (FFC), metronidazole (MTZ), neomycin (NEO), and sulfamethazine (SMZ) were respectively mixed with the same volume of Luminol-Eu-NCDs solution and placed in a quartz test tube for detection. Then, the fluorescence intensities of different mixed solutions were measured to determine the selectivity of Luminol-Eu-NCDs for NOR.

[0053] The results are as Figure 6 shown in d. These interfering antibiotics have no significant effect on the detection of Luminol-Eu-NCDs, demonstrating the high selectivity of Luminol-Eu-NCDs for NOR detection.

[0054] Application example To investigate the applicability and effectiveness of the Luminol-Eu-NCDs ratiometric fluorescence sensor for detecting NOR in real samples, milk samples were purchased from a nearby supermarket, and the pretreatment method was carried out according to the Chinese national standard (GB 5009.267-2020). Specifically: 0.5 mL of 1.0 mol / L zinc acetate solution and 0.5 mL of 0.3 mol / L potassium ferrocyanide were added to 2 mL of milk sample. After thorough mixing, the solution was centrifuged at 8000×g for 10 min. Subsequently, the supernatant was filtered through a 0.22 μm filter. NOR at 5 μmol / L, 40 μmol / L, and 80 μmol / L was dissolved in the supernatant. Then, 0.5 mL of the supernatant containing NOR was mixed with 0.5 mL of the Luminol-Eu-NCDs solution, and finally, its fluorescence intensity was measured on a fluorescence spectrophotometer.

[0055] Since NOR was not detected in the actual milk samples, the standard addition method was used to simulate the actual test. As shown in Table 1: Table 1 Application of the established method in the sensitive detection of NOR in milk samples The spiked recoveries of various spiked NOR in milk samples were 93.5% - 96.8%, and the relative standard deviations (RSDs) were 1.6% - 3.1%, indicating the reliability and acceptability of the Luminol-Eu-NCDs fluorescence sensor for detecting NOR in real samples. The results were basically consistent with those of the HPLC method. These data indicate that the ratiometric fluorescence sensor based on Luminol-Eu-NCDs is satisfactory for determining trace NOR in milk.

[0056] In summary, a ratiometric fluorescence probe Luminol-Eu-NCDs was synthesized by covalently linking Luminol-Eu-NPs and NCDs. The dual-signal response readout provides remarkable robustness, sensitivity, and selectivity for this method. In addition, the ratiometric method uses the fluorescence intensity at 430 nm as the internal reference and the fluorescence intensity at 542 nm as the detection signal, effectively reducing the influence of environmental fluctuations and enhancing its applicability in practical applications. The ratiometric fluorescence sensor based on Luminol-Eu-NCDs has a rapid fluorescence quenching response, a wide detection range, and a fast response time, and can detect NOR with acceptable selectivity and sensitivity. The ratiometric fluorescence sensor based on Luminol-Eu-NCDs can also quantitatively detect NOR in milk samples with satisfactory recoveries. This patent establishes a reliable, rapid, and accurate method for determining NOR residues in foods and the environment.

[0057] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a Luminol-Eu-NCDs ratio fluorescent probe, characterized in that: Here are the steps: (1) Luminol and Eu(NO3)3·6H2O were dissolved in Tris-HCl buffer and reacted under magnetic stirring to obtain Luminol-Eu; (2) NCDs were synthesized by solvothermal method using Listeria monocytogenes and urea as precursors and N,N-dimethylacetamide as reaction medium; (3) The Luminol-Eu prepared in step (1) and the NCDs prepared in step (2) are mixed in a solvent, stirred, centrifuged, and the supernatant is removed to obtain the Luminol-Eu-NCDs ratio fluorescence probe.

2. The method for preparing the Luminol-Eu-NCDs ratio fluorescent probe according to claim 1, characterized in that: In the step (1), the molar ratio of luminol to Eu(NO3)3·6H2O is 1:6-10; and the pH of the Tris-HCl buffer is 6-10.

3. The method for preparing the Luminol-Eu-NCDs ratio fluorescent probe according to claim 2, characterized in that: The temperature of the magnetic stirring is 20-40°C and the time is 20-50 min.

4. The method for preparing the Luminol-Eu-NCDs ratio fluorescent probe according to claim 1, characterized in that: The specific operations of step (2) are: ① Listeria monocytogenes was cultured to a concentration of 10 8 -10 9 CFU / mL, centrifuged and washed to obtain Listeria monocytogenes precipitate; ② adding urea to the obtained Listeria monocytogenes precipitate, and then resuspending it in N, N-dimethylacetamide to obtain a suspension; ③ The suspension was reacted at 160-200 °C for 8-12 h to obtain a dark brown solution, which was then centrifuged, filtered through a 0.22 μm filter membrane, purified, and rotary evaporated to obtain NCDs.

5. The method for preparing Luminol-Eu-NCDs according to claim 1, characterized in that: In the step (3), the mass ratio of Luminol-Eu to NCDs is 280-320:1; and the solvent is water or ethanol.

6. The method for preparing Luminol-Eu-NCDs according to claim 5, characterized in that: The mixing and stirring in step (3) is performed at a temperature of 20-40° C. and for a time of 20-50 min.

7. Luminol-Eu-NCDs ratiometric fluorescent probe prepared by the method described in any one of claims 1 to 6.

8. Use of the Luminol-Eu-NCDs ratiometric fluorescent probe according to claim 7 in detecting norfloxacin for purposes other than disease diagnosis.

9. A method for detecting norfloxacin for purposes other than disease diagnosis, characterized in that: The steps are as follows: add equal volume ratios of Luminol-Eu-NCDs ratio fluorescent probe solution and the sample to be detected into the cuvette in sequence, record the fluorescence intensity ratio at 542 nm and 430 nm as y after stabilization, substitute it into the linear equation y=-0.0568x+6.73812, and calculate the concentration x of norfloxacin in the sample to be detected.

10. The method for detecting norfloxacin for non-disease diagnosis purposes according to claim 9, characterized in that: The concentration of the Luminol-Eu-NCDs ratio fluorescent probe solution is 4-8 mg / mL; The samples to be tested are pre-processed foods.

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